Conformationally constrained parathyroid hormone (pth) analogs

ABSTRACT

The present invention relates to conformationally constrained parathyroid hormone (PTH) analogs, and methods of preparing and using the PTH analogs. The invention provides novel PTH polypeptide derivatives containing amino acid substitutions at selected positions in the polypeptides. The invention provides derivatives of PTH (1-34), PTH(1-21), PTH(1-20), PTH(1-19), PTH(1-18), PTH(1-17), PTH(1-16), PTH(1-15), PTH(1-14), PTH(1-13), PTH(1-12), PTH(1-11) and PTH(1-1 0) polypeptides, wherein at least one residue in each polypeptide is a helix, preferably an a-helix, stabilizing residue. The invention also provides methods of making such peptides. Further, the invention encompasses compositions and methods for use in limiting undesired bone loss in a vertebrate at risk of such bone loss, in treating conditions that are characterized by undesired bone loss or by the need for bone growth, e.g. in treating fractures or cartilage disorders and for raising cAMP levels in cells where deemed necessary.

Background of the Invention

1. Field of the Invention

The present invention relates to conformationally constrainedparathyroid hormone (PTH) analogs, and methods of preparing and usingthe PTH analogs.

2. Background Art

Parathyroid Hormone

Parathyroid hormone (PTH), an 84 amino acid peptide, is the principalregulator of ionized blood calcium in the human body (Kronenberg, H. M.,et al., In Handbook of Experimental Pharmacology, Mundy, G. R., andMartin, T. J., (eds), pp. 185-201, Springer-Verlag, Heidelberg (1993)).Regulation of calcium concentration is necessary for the normal functionof the gastrointestinal, skeletal, neurologic, neuromuscular, andcardiovascular systems. PTH synthesis and release are controlledprincipally by the serum calcium level; a low level stimulates and ahigh level suppresses both hormone synthesis and release. PTH, in turn,maintains the serum calcium level by directly or indirectly promotingcalcium entry into the blood at three sites of calcium exchange: gut,bone, and kidney. PTH contributes to net gastrointestinal absorption ofcalcium by favoring the renal synthesis of the active form of vitamin D.PTH promotes calcium resorption from bone indirectly by stimulatingdifferentiation of the bone-resorbing cells, osteoclasts. It alsomediates at least three main effects on the kidney: stimulation oftubular calcium reabsorption, enhancement of phosphate clearance, andpromotion of an increase in the enzyme that completes synthesis of theactive form of vitamin D. PTH is thought to exert these effectsprimarily through receptor-mediated activation of adenylate cyclaseand/or phospholipase C.

Disruption of calcium homeostasis may produce many clinical disorders(e.g., severe bone disease, anemia, renal impairment, ulcers, myopathy,and neuropathy) and usually results from conditions that produce analteration in the level of parathyroid hormone. Hypercalcemia is acondition that is characterized by an elevation in the serum calciumlevel. It is often associated with primary hyperparathyroidism in whichan excess of PTH production occurs as a result of a parathyroid glandlesion (e.g., adenoma, hyperplasia, or carcinoma). Another type ofhypercalcemia, humoral hypercalcemia of malignancy (HHM) is the mostcommon paraneoplastic syndrome. It appears to result in most instancesfrom the production by tumors (e.g., squamous, renal, ovarian, orbladder carcinomas) of a class of protein hormone which shares aminoacid homology with PTH. These PTH-related proteins (PTHrP) appear tomimic certain of the renal and skeletal actions of PTH and are believedto interact with the PTH receptor in these tissues.

Osteoporosis

Osteoporosis is a potentially crippling skeletal disease observed in asubstantial portion of the senior adult population, in pregnant womenand even in juveniles. The term osteoporosis refers to a heterogeneousgroup of disorders. Clinically, osteoporosis is separated into type Iand type II. Type I osteoporosis occurs predominantly in middle agedwomen and is associated with estrogen loss at menopause, whileosteoporosis type II is associated with advancing age. Patients withosteoporosis would benefit from new therapies designed to promotefracture repair, or from therapies designed t6 prevent or lessen thefractures associated with the disease.

The disease is marked by diminished bone mass, decreased bone mineraldensity (BMD), decreased bone strength and an increased risk of bonefracture. At present, there is no effective cure for osteoporosis,though estrogen, calcitonin and the bisphosphonates, etidronate andalendronate are used to treat the disease with varying levels ofsuccess. These agents act to decrease bone resorption. Since parathyroidhormone regulates blood calcium and the phosphate levels, and has potentanabolic (bone-forming) effects on the skeleton, in animals (Shen, V.,et al., Calcif Tissue Int. 50:214-220 (1992); Whitefild, J. F., et al.,Calcif Tissue Int. 56:227-231 (1995) and Whitfield, J. F., et al.,Calcif Tissue Int. 60:26-29 (1997)) and humans (Slovik, D. M., et al.,J. Bone Miner. Res. 1:377-381 (1986); Dempster, D. W., et al., Endocr.Rev. 14:690-709 (1993) and Dempster, D. W., et al., Endocr. Rev. 15:261(1994)) when administered intermittently, PTH, or PTH derivatives, areprime candidates for new and effective therapies for osteoporosis.

PTH Derivatives

PTH derivatives include polypeptides that have amino acid substitutionsor are truncated relative to the full length molecule. Both a 14 and a34 amino acid amino-terminal truncated form of PTH, as well as aC-terminal truncated form have been studied. Additionally, amino acidsubstitutions within the truncated polypeptides have also beeninvestigated.

Synthetic PTH(1-34) exhibits full bioactivity in most cell-based assaysystems, has potent anabolic effects on bone mass in animals and hasrecently been shown to reduce the risk of bone fracture inpostmenopausal osteoporotic women (Neer, R. M., et al., N.E.J.M.344:1434-1441 (2001); Dempster, D. W., et al., Endocr Rev 14:690-709(1993)). PTH acts on the PTH/PTHrP receptor (P1R), a class II Gprotein-coupled heptahelical receptor that couples to the adenylylcyclase/CAMP and phospolipase C/inositol phosphate (IP) signalingpathway (Rippner, H., et al., Science 254:1024-1026 (1991)). Deletionanalysis studies have shown that the amino-terminal residues of PTH playa crucial role in stimulating the P1 R to activate the cAMP and IPsignaling pathways (Tregear, G. W., et al., Endocrinology 93:1349-1353(1973); Takasu, H., et al., Biochemistry 38:13453-13460(1999)).Crosslinking and receptor mutagenesis studies have indicated thatresidues in the amino-terminal portion of PTH interact with theextracellular loops and extracellular ends of the seven transmembranehelices, which reside within the juxtamembrane region of the receptor(Bergwitz, C., et al., J. Biol. Chem. 271:26469-26472 (1996); Hoare, S.R. J., et al., J. Biol. Chem 276:7741-7753 (2001); Behar, V., et al., J.Biol. Chem. 275:9-17 (1999); Shimizu, M., et al., J. Biol. Chem.275:19456-19460(2000); Luck, M. D., et al., Molecular Endocrinology13:670-680 (1999)).

BRIEF SUMMARY OF THE INVENTION

The invention provides novel PTH polypeptide derivatives containingamino acid substitutions at selected positions in the polypeptides. Thederivatives function as full, or nearly full, agonists of the PTH-1receptor. Because of their unique properties, these polypeptides have autility as drugs for treating human diseases of the skeleton, such asosteoporosis.

The invention provides derivatives of PTH(1-21), PTH(1-20), PTH(1-19),PTH(1-18),PTH(1-17), PTH(1-16), PTH(1-15), PTH(1-14), PH(1-13),PTH(1-12), PTH(1-11) and PTH(1-10) polypeptides, wherein at least oneresidue in each polypeptide is a helix, preferably an α-helix,stabilizing residue. The invention also provides methods of making suchpeptides. Further, the invention encompasses compositions and methodsfor use in limiting undesired bone loss in a vertebrate at risk of suchbone loss, in treating conditions that are characterized by undesiredbone loss or by the need for bone growth, e.g. in treating fractures orcartilage disorders and for raising cAMP levels in cells where deemednecessary.

In one aspect, the invention is directed to a biologically activepeptide consisting essentially of X₀₁ValX₀₂GluIleGlnLeuMetHisX₀₃X₀₄X₀₅X₀₆X₀₇ (SEQ ID NO: 1), wherein X₀₁ isan α-helix-stabilizing residue, desaminoGly, desaminoSer or desaminoAla;X₀₂ is an α-helix-stabilizing residue, Ala, or Ser; X₀₃ is Ala, Gln orAsn; X₀₄ is Arg, Har or Leu; X₀₅ is an α-helix-stabilizing residue, Alaor Gly; X₀₆ is an α-helix-stabilizing residue or Lys; and X₀₇ is anα-helix-stabilizing residue, Trp or His: and wherein at least one ofX₀₁, X₀₂, X₀₃, X₀₄, X₀₅, X₀₆ or X₀₇ is an α-helix-stabilizing residue.

In another aspect, the invention relates to SEQ ID NO: 1, wherein theα-helix-stabilizing amino acid is selected from the group consisting ofAib, ACPC (1-aminocyclopropylcarboxylic acid), DEG (diethylglycine) and1-aminocyclopentanecarboxylic acid. In another aspect, the inventionrelates to SEQ ID NO: 1, wherein the α-helix-stabilizing amino acid isAib.

The invention is further drawn to fragments ofthe peptide of SEQ ID NO:1, in particular X₀₁ ValX₀₂GluIleGlnLeuMetHisX₀₃X₀₄X₀₅X₀₆ (SEQ ID NO:12), X₀₁ ValX₀₂GluIleGlnLeuMetHisX₀₃X₀₄X₀₅ (SEQ ID NO: 13), X₀₁ValX₀₂GluIleGlnLeuMetHisX₀₃X₀₄ (SEQ ID NO: 14) and X₀₁ValX₀₂GluIleGlnLeuMetHisX₀₃ (SEQ ID NO: 15). The invention furtherencompasses pharmaceutically acceptable salts of the above-describedpeptides, and N- or C-derivatives of the peptides. A preferableembodiment of the invention is drawn to any of the above recitedpolypeptides, wherein the polypeptide contains a C-terminal amnide.

In addition, the invention is drawn to a biologically active polypeptideconsisting essentially ofAibValAibGluIleGlnLeuNleHisGlnHarAlaLysTrpLeu-AlaSerValArgArtTyr (SEQ IDNO. 8); fragments thereof, containing amino acids 1-20, 1-19, 1-18,1-17, 1-16 or 1-15; pharmaceutically acceptable salts thereof; or N- orC-derivatives thereof.

The invention is further drawn to any of the above polypeptides labeledwith a label selected from the group consisting of: a radiolabel, aflourescent label, a bioluminescent label, or a chemiluminescent label.In a preferable embodiment the radiolabel is ¹²⁵I or ^(99m)Tc.

Preferred embodiments of the biologically active peptide include:AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 2);desamino-AlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 3);desamino-SerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 4);desamino-GlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 5);AibValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 6);AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 7);AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 9);AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 10);SerValAibGluIleGffi uMetHisGlnHarAlaLysTrp (SEQ ID NO. 11); andAibValAibGluIleGlnLeuMetHisGlnHar (SEQ ID NO. 16). It is contemplatedthat fragments of the above mentioned peptides, containing amino acids1-10, 1-11, 1-12 or 1-13, are also embodiments of the present invention.The invention further encompasses pharmaceutically acceptable salts ofthe above-described peptides, and N- or C-derivatives of the peptides.

Other constrained amino acids that are substituted for Aib are ACPC(1-aminocyclopropylcarboxylic acid), DEG (diethylglycine) and1-aminocyclopentanecarboxylic acid.

In accordance with yet a further aspect of the invention, this inventionalso provides pharmaceutical compositions comprising a PTH derivativeand a pharmaceutically acceptable excipient and/or a pharmaceuticallyacceptable solution such as saline or a physiologically bufferedsolution.

This invention also provides a method for treating mammalian conditionscharacterized by decreases in bone mass, which method comprisesadministering to a subject in need thereof an effective bonemass-increasing amount of a biologically active PTH polypeptide. Apreferable embodiment of the invention is drawn to conditions such asosteoporosis. The types of osteoporosis include, but are not limited toold age osteoporosis and postmenopausal osteoporosis. Additionalpreferable embodiments include using an effective amounts of thepolypeptide of about 0.01 μg/kg/day to about 1.0 μg/kg/day wherein thepolypeptide is administered parenterally, subcutaneously or by nasalinsufflation.

In accordance with yet a further aspect of the invention, this inventionalso provides a method for determining rates of bone reformation, boneresorption and/or bone remodeling comprising administering to a patientan effective amount of a labeled PTH polypeptide, such as for example,SEQ ID NO: 1 or a derivatives thereof and determining the uptake of thepeptide into the bone of the patient. The peptide is labeled with alabel selected from the group consisting of: radiolabel, flourescentlabel, bioluminescent label, or chemiluminescent label. An example of asuitable radiolabel is ^(99m)Tc.

The invention is further related to a method of increasing cAMP in amammalian cell having PTH-1 receptors, the method comprising contactingthe cell with a sufficient amount of the polypeptide of the invention toincrease cAMP.

The invention also provides derivatives of rat PTH(1-34) (rPTH(1-34))given by AlaValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAlaSerValGluArgMetGlnTrpLeuArgLysLysLeuGlnAspValHisAsnPhe (SEQ ID NO: 30), and of humanPTH(1-34) (hPTH(1-34)) given by SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsnSerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHisAsnPhe (SEQ ID NO: 31).

In another aspect, the invention relates to a biologically activepeptide consisting essentially ofthe formula X₀₁ValX₀₂GluIleGlnLeuX₀₃HisX₀₄X₀₅X₀₆X₀₇X₀₈LeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄(SEQ ID NO: 19) wherein X₀₁ is an α-helix-stabilizing residue,desaminoGly, desaminoSer or desaminoAla; X₀₂ is an α-helix-stabilizingresidue, Ala, or Ser; X₀₃ is Met or Nle; X₀₄ is Ala, Gln or Asn; X₀₅ isArg, Har or Leu; X₀₆ is an α-helix-stabilizing residue, Ala or Gly; X₀₇is an α-helix-stabilizing residue or Lys; X₀₈ is an α-helix-stabilizingresidue, Trp or His; X₀₉ is Ala or Asn; X₁₀ is Met or Val; X₁₁ is Arg orGlu; X₁₂ is Met or Val; X₁₃ is Gln or Glu; X₁₄ is Tyr or Phe; andwherein at least one of X₀₁, X₀₂, X₀₆, X₇, or X₀₈ is anα-helix-stabilizing residue. The invention also relates to fragmentsthereof, containing amino acids 1-33, 1-32, 1-31, 1-30, 1-29, 1-28,1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16,1-15, 1-14, 1-13, 1-12, or 1-11. The invention also relates topharmaceutically acceptable salts and N- or C-derivatives of SEQ ID NO:19 or the above described fragments.

In another aspect, the invention relates to SEQ ID NO: 19, wherein theα-helix-stabilizing amino acid is selected from the group consisting ofAib, ACPC (1-aminocyclopropylcarboxylic acid), DEG (diethylglycine) and1-aminocyclopropylcarboxylic acid. In another aspect, the inventionrelates to SEQ ID NO: 19, wherein the α-helix-stabilizing amino acid isAib.

In another aspect, the invention relates specifically to the followingpeptides: AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉Ser₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 20);desaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ IDNO. 21); desaminoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ IDNO. 22); desaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO.23); AibValAibGluIleGlnLeuMetHisGlnHarGlyLysTrpLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 24);AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉Ser X₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 25);AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 26);AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsn X₁₄ (SEQ ID NO. 27); andSerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeu X₀₉Ser X₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 28). X₀₉, X₁₀, X₁₁, X₁₂,X₁₃ and X₁₄ have the same meaning as defined for SEQ ID NO: 19. Theinvention also relates to pharmaceutically acceptable salts or N- orC-derivatives of the above peptides.

The invention also relates to a biologically active peptide consistingessentially of the formula AibValAibGluIleGlnLeuNleHisGlnHarAlaLysTrpLeuAlaSerValArgArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO:29) wherein X₁₂ is Met or Val; X₁₃ is Gln or Glu; and X₁₄ is Tyr or Phe.The invention also relates to pharmaceutically acceptable salts or N- orC-derivatives of SEQ ID NO: 29. The invention also relates to fragmentsthereof, containing amino acids 1-33, 1-32, 1-31, 1-30, 1-29, 1-28,1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16,1-15, 1-14, 1-13, 1-12, or 1-11.

In another aspect of the invention, SEQ ID NO: 19, SEQ ID NO: 29 or anyof the above peptides are labeled. In another aspect of theinvention,SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides arelabeled with a fluorescent label, a chemiluminescent label; abioluminescent label; a radioactive label; ¹²⁵I; or ^(99m)Tc.

In another aspect, the invention is directed to a pharmaceuticalcomposition comprising the biologically active peptide SEQ ID NO: 19,SEQ ID NO: 29 or any of the above peptides, and a pharmaceuticallyacceptable carrier.

In another aspcet, the invention is directed to a method for treatingmammalian conditions characterized by decreases in bone mass, the methodcomprising administering to a subject in need thereof an effective bonemass-increasing amount of a biologically active peptide of SEQ ID NO:19, SEQ ID NO: 29 or any of the above peptides.

In another aspcet, the invention is directed to a method for treatingmammalian conditions characterized by decreases in bone mass, the methodcomprising administering to a subject in need thereof an effective bonemass-increasing amount of a composition comprising a biologically activepeptide of SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides anda pharmaceutically acceptable carrier.

In another aspect of the invention, the condition to be treated isosteoporosis, old age osteoporosis, or post-menopausal osteoporosis. Inanother aspect of the invention, the effective amount of SEQ ID NO: 19,SEQ ID NO: 29 or any of the above peptides for increasing bone mass isfrom about 0.01 μg/kg/day to about 1.0 μg/kg/day. In another aspect ofthe invention, the method of administration is parenteral, subcutaneousor nasal insufflation.

In another aspcet, the invention is directed to a method for determiningrates of bone reformation, bone resorption and/or bone remodelingcomprising administering to a patient an effective amount of SEQ ID NO:19, SEQ ID NO: 29 or any of the above peptides and determining theuptake of the peptide into the bone of the patient.

In another aspcet, the invention is directed to a method of making SEQID NO: 19, SEQ ID NO: 29 or any of the above peptides, wherein thepeptide is synthesized by solid phase synthesis. in another aspcet, theinvention is directed to a method of making SEQ ID NO: 19, SEQ ID NO: 29or any of the above peptides, wherein the peptide is protected by FMOC.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Aib-scan of a modified PTH(1-14) analog in HKRK-B28 cells. Thepeptide [Ala^(3,12),Gln¹⁰,Har¹¹,Trp¹⁴]PTH(1-14) amide {[M]PTH(1-14)},and derivatives of that peptide containing a single Aib substitution atone of each residue position, were evaluated for the capacity tostimulate intracellular cAMP accumulation in HKRK-B28 cells. Thepeptides with substitutions at position 1-7 are shown in panel A, andthose with substitutions at position 8-9 are shown in B. Shown arecombined data (mean±S.E.M.) from 3 to 10 experiments, each performed induplicate. Symbols are defined in the key.

FIG. 2. cAMP-signaling and binding properties of PTH analogs in HKRK-B28cells. Peptides were evaluated in HKRK-B28 cells for the capacity tostimulate intracellular cAMP accumulation (A) and the capacity toinhibit binding of ¹²⁵I-[M]PTH(1-21) (B). Shown are combined data(mean±S.E.M.) from 3 or 4 experiments, each performed in duplicate.Peptides and corresponding symbols are identified in the key.

FIG. 3. Signaling and binding properties of PTH analogs in COS-7 cellsexpressing an N-terminally truncated P1R. COS-7 cells were transientlytransfectd with P1R-delNt, a truncated P1R which is deleted for most ofthe amino-terminal extracellular domain, and subsequently used toevaluate the capacities of the indicated PTH analogs to stimulateintracellular cAMP accumulation (A); stimulate formation of ³H-inositolphosphates (IP¹+IP²+IP³) (B); and inhibit the binding of¹²⁵I-[Aib^(1,3),M]PTH(1-21) (C). Each curve shows data combined(mean±S.E.M.) from 3 to 6 experiments, each performed in duplicate. Themean basal level of³H-inositol phosphates (2,929±877 cpm/well) isindicated by the dashed iine. Peptides and corresponding symbols areidentified in the key.

FIG. 4. cAMP-signaling properties of PTH analogs in SaOS-2 cells. Thepeptides, PTH(1-34), native PTH(1-14), [M]PTH(1-14) and[Aib^(1,3),M]PTH(1-14) were evaluated in the human osteosarcoma-derivedcell line SaOS-2 for the capacity to stimulate intracellular cAMPaccumulation. Shown are combined data (mean±S.E.M.) from 3 or 4experiments, each performed in duplicate. Symbols are defined in theKey.

FIG. 5. Effect of PTH analogs on bone mineralization in embryonic mousemetatarsals. Cartilaginous metatarsal bone rudiments were excised fromE15.5 mouse embryos and transferred to tissue culture plates containingserum-free media. Added to the samples for 48 h were vehicle: (A);PTH(1-34) (0.1 μM) (B); [Aib^(1,3),M]PTH(1-14) (1 μM) (C) or nativePTH(1-14) (2 μM) (D). Samples were explanted and incubated at 37° C. fora total of 64 h; peptide or vehicle were added at 16 h and again at 24h. At the end of the incubation, the samples were fixed, sectioned anddirectly visualized under white light using a dissecting scope. In thevehicle- and native PTH(1-14)-treated samples mineralization can bedetected as dark material at the center of the bone rudiment. BothPTH(1-34) and [Aib^(1,3),M]PTH(1-14) inhibited mineralization. Shown aredata from a single experiment, comparable results were obtained in threeother replicate experiments.

FIG. 6. Circular Dichroism Spectroscopy. Spectra were recorded for theindicated N-terminal PTH oligopeptides, each at 20 μM, in 50 nM sodiumphosphate buffer, pH 7.4 containing 20% 2,2,2,-trifluoroethanol. Thenegative extrema in the spectra at ˜209 nM and ˜222 nM, and the positiveextrema at ˜192 nM, which are more apparent in the Aib-containing PTHanalogs, as compared to the non-Aib-containing peptides, are indicativeof helical content.

FIG. 7. Signaling and binding properties of hPTH(1-34) analogs in COS-7cells expressing wildtype P1R (hP1R-WT, FIG. 7A) and N-terminallytruncated P1R (hP1R-delNT, FIG. 7B). The COS-7 cells were used toevaluate the capacities of the indicated PTH analogs to stimulateintracellular cAMP accumulation. Cells expressing hP1R-delNT wereprepared as described above. Peptides and corresponding symbols areidentified in the key.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Amino Acid Sequences: The amino acid sequences in this application useeither the single letter or three letter designations for the aminoacids. These designations are well known to one of skill in the art andcan be found in numerous readily available references, such as forexample in Cooper, G. M., The Cell 1997, ASM Press, Washington, D.C. orAusubel et al., Current Protocols in Molecular Biology, 1994. Wheresubstitutions in a sequence are referred to, for example, as Ser-3→Alaor [Ala³]peptide, this means that the serine in the third position fromthe N-terminal end of the polypeptide is replaced with another aminoacid, Alanine in this instance.

In the present application [M]PTH(1-14) is defined as[Ala^(3,12),Gln¹⁰,Har¹¹,Trp¹⁴]PTH(1-14)amide. [M]PTH(1-21)is defined as[Ala^(3,12),Nle⁸,Gln¹⁰,Har¹¹,Trp¹⁴,Arg¹⁹,Tyr²¹]PTH(1-21)amide.[M]PTH(1-11) is defined as [Ala³,Gln¹⁰,Har¹¹]PTH(1-11)amide.

In the present application, “Aib” refers to α-aminoisobutyric acid;“Har” refers to homoarginine; “Nle” refers to norleucine; and otheramino acids are in either the conventional one- or three-letter codes.

Biological Activity of the Protein: This expression refers to anybiological activity of the polypeptide. Examples of these activitiesinclude, but are not limited to metabolic or physiologic function ofcompounds of SEQ ID NO: 1 or SEQ ID NO: 8 or derivatives thereof,including similar activities or improved activities, or those activitieswith decreased undesirable side-effects. Also included are antigenic andimmunogenic activities of the above-described compounds.

Derivative or Functional Derivative: The term “derivative” or“functional derivative” is intended to include “variants,” the“derivatives,” or “chemical derivatives” of the PTH molecule. A“variant” of a molecule such as for example, a compound of SEQ ID NO: 1or derivative thereof is meant to refer to a molecule substantiallysimilar to either the entire molecule, or a fragment thereof. An“analog” of a molecule such as for example, a compound of SEQ ID NO: 1orderivative thereof is meant to refer to a non-natural moleculesubstantially similar to either the SEQ ID NO: 1 molecules or fragmentsthereof.

PTH derivatives contain changes in the polypeptide relative to thenative PTH polypeptide of the same size. The sequence of the nativePTH(1-14) polypeptide is the first fourteen amino acids of SEQ. ID NO:17 (human PTH (1-21))or SEQ. ID NO: 18(rat PTH(1-21)). A molecule issaid to be “substantially similar” to another molecule if the sequenceof amino acids in both molecules is substantially the same, and if bothmolecules possess a similar biological activity. Thus, two moleculesthat possess a similar activity, may be considered variants,derivatives, or analogs as that term is used herein even if one of themolecules contains additional amino acid residues not found in theother, or if the sequence of amino acid residues is not identical. PTHderivatives, however, need not have substantially similar biologicalactivity to the native molecule. In some instances PTH derivatives havesubstantially different activity than the native PTH. For example, aderivative may be either an antagonist or an agonist of the PTHreceptor.

As used herein, a molecule is said to be a “chemical derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties may improve themolecule's solubility, absorption, biological half-life, etc. Themoieties may alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Examples of moieties capable of mediating such effects are disclosed inRemington's Pharmaceutical Sciences (1980) and will be apparent to thoseof ordinary skill in the art.

Fragment: A “fragment” of a molecule such as for example, SEQ ID NO: 1or derivative thereof is meant to refer to any polypeptide subset ofthese molecules.

Fusion protein: By the term “fusion protein” is intended a fused proteincomprising compounds such as for example, SEQ ID NO: 1 or derivativesthereof, either with or without a “selective cleavage site” linked atits N-terminus, which is in turn linked to an additional amino acidleader polypeptide sequence.

Polypeptide: Polypeptide and peptide are used interchangeably. The termpolypeptide refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids andinclude amino acid sequences modified either by natural processes, suchas post-translational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in theresearch literature. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications.

Polypeptides may be branched and they may be cyclic, with or withoutbranching. Cyclic, branched and branched cyclic polypeptides may resultfrom post-translational modifications or may be made by syntheticmethods. Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination. See, for instance,Proteins-Structure and Molecular Properties, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993 and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press. New York, 1983; Seifter et al., “Analysisfor protein modifications and nonprotein cofactors”, Methods in Enzymol.182:626-646 (1990) and Rattan et al., “Protein Synthesis:Posttranslational Modifications and Aging”, Ann NY Acad Sci 663:48-62(1992).

PTH Analogs—Structural and Functional Properties

α-aminoisobutyric acid (Aib) was introduced into short N-terminal PTHpeptide analogs. The numerous NMR studies of PTH(1-34) analogs,performed in a variety of polar or non-polar solvents, have generallyindicated two domains of secondary structure: a stable C-terminal helixextending approximately from Ser-17 to Val-31, and a shorter and lessstable amino-terminal helix, extending variably from Ser-3 to Lys-13,the two domain being connected by a bend or turn region (Marx, U. C., etal., Biochem. Biophys. Res. Commun. 267:213-220 (2000); Chen, Z., etal., Biochemistry 39:12766-12777 (2000); Marx, U. C., et al., J. BiolChem. 270:15194-15202 (1995); Marx, U. C., et al., J. Biol. Chem.273:4308-4316 (1998); Pellegrini, M., et al., Biochemistry37:12737-12743 (1998); Gronwald, W., et al., Biol. Chem. Hoppe Seyler377:175-186 (1996); Barden, J. A., and Kemp, B. E., Biochemistry32:7126-7132 (1993)). The recent crystallographic study of PTH(1-34)indicated a continuous α-helix extending from Ser-3 to His-32 andcontaining only a slight 15° bend at the midsection. However, NMR dataindicates that the N-terminal α-helix is relatively weak.Helix-stabilizing modifications, such as the introduction of Aibresidues, offer significant benefits in terms of peptide potency, andresult in short peptides (≦14 amino acids) with activity comparable toPTH(1-34).

Described herein are novel “minimized” variants of PTH that are smallenough to be deliverable by simple non-injection methods. The variantsof the present invention contain substitutions in the first 14 aminoacids of the polypeptide. The new polypeptides correspond to the 1-21,1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, and 1-10amino acid sequence of the mature PTH polypeptide. The shorter variants(≦PTH1-14) have a molecular weight of less than 2,000 daltons.

The primary amino acid sequence of the native human PTH(1-21) peptide(N-terminus to C-terminus) isSerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsnSerMetGluArgVal (SEQ IDNO: 17), whereas the primary sequence of the native rat PTH (1-21) isAlaValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAlaSerValGluArgMet (SEQ IDNO. 18).

As protein products, compounds described herein are amenable toproduction by the techniques of solution- or solid-phase peptidesynthesis. The solid phase peptide synthesis technique, in particular,has been successfully applied in the production of human PTH and can beused for the production of these compounds (for guidance, see Kimura etal., supra, and see Fairwell et al., Biochem. 22:2691 (1983)). Successwith producing human PTH on a relatively large scale has been reportedby Goud et al., in J. Bone Min. Res. 6(8):781 (1991). The syntheticpeptide synthesis approach generally entails the use of automatedsynthesizers and appropriate resin as solid phase, to which is attachedthe C-terminal amino acid of the desired compounds of SEQ ID NO: 1 orderivatives thereof. Extension of the peptide in the N-terminaldirection is then achieved by successively coupling a suitably protectedform of the next desired amino acid, using either FMOC- or BOC-basedchemical protocols typically, until synthesis is complete. Protectinggroups are then cleaved from the peptide, usually simultaneously withcleavage of peptide from the resin, and the peptide is then isolated andpurified using conventional techniques, such as by reversed phase HPLCusing acetonitrile as solvent and tri-fluoroacetic acid as ion-pairingagent. Such procedures are generally described in numerous publicationsand reference may be made, for example, to Stewart and Young, “SolidPhase Peptide Synthesis,” 2nd Edition, Pierce Chemical Company,Rockford, Ill. (1984). It will be appreciated that the peptide synthesisapproach is required for production of such as for example, SEQ ID NO: 1and derivatives thereof which incorporate amino acids that are notgenetically encoded, such as Aib.

In accordance with another aspect of the present invention, substituentsare attached to the free amine of the N-terminal amino acid of compoundsof the present invention standard methods known in the art. For example,alkyl groups, e.g., C₁₋₁₂ alkyl, are attached using reductivealkylation. Hydroxyalkyl groups, e.g. C₁₋₁₂ hydroxyalkyl, are alsoattached using reductive alkylation wherein the free hydroxy group isprotected with a t-butyl ester. Acyl groups, e.g., COE₁, are attached bycoupling the free acid, e.g., E₁COOH, to the free amino of theN-terminal amino acid. Additionally, possible chemical modifications ofthe C-terminal end of the polypeptide are encompassed within the scopeof the invention. These modifications may modify binding affinity to thereceptor.

Also contemplated within the scope of this invention are those compoundssuch as for example, SEQ ID NO: 1 and derivatives thereof with alteredsecondary or tertiary structure, and/or altered stability, which stillretain biological activity. Such derivatives might be achieved throughlactam cyclization, disulfide bonds, or other means known to a person ofordinary skill in the art.

Utility and Administration of Compounds of the Invention

Compounds of the invention or derivatives thereof have multiple uses.These include, inter alia, agonists or antagonists ofthe PTH receptor,prevention and treatment of a variety of mammalian conditions manifestedby loss of bone mass, diagnostic probes, antigens to prepare antibodiesfor use as diagnostic probes and even as molecular weight markers. Beingable to specifically substitute one or more amino acids in the PTHpolypeptide permits construction of specific molecular weightpolypeptides.

In particular, the compounds of this invention are indicated for theprophylaxis and therapeutic treatment of osteoporosis and osteopenia inhumans. Furthermore, the compounds of this invention are indicated forthe prophylaxis and therapeutic treatment of other bone diseases. Thecompounds of this invention are also indicated for the prophylaxis andtherapeutic treatment of hypoparathyroidism. Finally, the compounds ofthis invention are indicated for use as agonists for fracture repair andas antagonists for hypercalcemia.

In general, compounds of the present invention, or salts thereof, areadministered in amounts between about 0.01 and 1 μg/kg body weight perday, preferably from about 0.07 to about 0.2 μg/kg body weight per day.For a 50 kg human female subject, the daily dose of biologically activecompound is from about 0.5 to about 50 μgs, preferably from about 3.5 toabout 10 μgs. In other mammals, such as horses, dogs, and cattle, higherdoses may be required. This dosage may be delivered in a conventionalpharmaceutical composition by a single administration, by multipleapplications, or via controlled release, as needed to achieve the mosteffective results, preferably one or more times daily by injection. Forexample, this dosage may be delivered in a conventional pharmaceuticalcomposition by nasal insufflation.

The selection ofthe exact dose and composition and the most appropriatedelivery regimen will be influenced by, inter alia, the pharmacologicalproperties of the selected compounds of the invention, the nature andseverity of the condition being treated, and the physical condition andmental acuity of the recipient.

Representative preferred delivery regimens include, without limitation,oral, parenteral, subcutaneous, transcutaneous, intramuscular andintravenous, rectal, buccal (including sublingual), transdermal, andintranasal insufflation.

Pharmaceutically acceptable salts retain the desired biological activityof the compounds of the invention without toxic side effects. Examplesof such salts are (a) acid addition salts formed with inorganic acids,for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; and salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid and the like; (b) base additionsalts formed with polyvalent metal cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium,and the like; or with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; or (c) combinations of(a) and (b), e.g., a zinc tannate salt and the like. Pharmaceuticallyacceptable buffers include but are not limited to saline or phosphatebuffered saline. Also included in these solutions may be acceptablepreservative known to those of skill in the art.

A further aspect of the present invention relates to pharmaceuticalcompositions comprising as an active ingredient compounds of theinvention or derivatives thereof of the present invention, orpharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable, non-toxic carrier. As mentioned above, suchcompositions may be prepared for parenteral (subcutaneous,transcutaneous, intramuscular or intravenous) administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for rectal, transdermal administration; and for intranasaladministration, particularly in the form of powders, nasal drops oraerosols.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985), incorporated herein by reference. Formulations for parenteraladministration may contain as excipients sterile water or saline,alkylene glycols such as propylene glycol, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. For oral administration, the formulation can be enhancedby the addition of bile salts or acylcarnitines. Formulations for nasaladministration may be solid and may contain excipients for example,lactose or dextran, or may be aqueous or oily solutions for use in theform of nasal drops or metered spray. For buccal administration typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

When formulated for the most preferred route of administration, nasaladministration, the absorption across the nasal mucous membrane may beenhanced by surfactant acids, such as for example, glycocholic acid,cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid,chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid,cyclodextrins and the like in an amount in the range between about 0.2and 15 weight percent, preferably between about 0.5 and 4 weightpercent, most preferably about 2 weight percent.

Delivery of the compounds of the present invention to the subject overprolonged periods of time, for example, for periods of one week to oneyear, may be accomplished by a single administration of a controlledrelease system containing sufficient active ingredient for the desiredrelease period. Various controlled release systems, such as monolithicor reservoir-type microcapsules, depot implants, osmotic pumps,vesicles, micelles, liposomes, transdermal patches, iontophoreticdevices and alternative injectable dosage forms may be utilized for thispurpose. Localization at the site to which delivery of the activeingredient is desired is an additional feature of some controlledrelease devices, which may prove beneficial in the treatment of certaindisorders.

One form of controlled release formulation contains the polypeptide orits salt dispersed or encapsulated in a slowly degrading, non-toxic,non-antigenic polymer such as copoly(lactic/glycolic) acid, as describedin the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No.4,675,189. The compounds or, preferably, their relatively insolublesalts, may also be formulated in cholesterol or other lipid matrixpellets, or silastomer matrix implants. Additional slow release, depotimplant or injectable formulations will be apparent to the skilledartisan. See, for example, Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New York,1978, and R. W. Baker, Controlled Release of Biologically Active Agents,John Wiley & Sons, New York, 1987.

Like PTH, the PTH variants may be administered in combination with otheragents usefull in treating a given clinical condition. When treatingosteoporosis and other bone-related disorders for example, the PTHvariants may be administered in conjunction with a dietary calciumsupplement or with a vitamin D analog (see U.S. Pat. No. 4,698,328).Alternatively, the PTH variant may be administered, preferably using acyclic therapeutic regimen, in combination with bisphosphonates, asdescribed for example in U.S. Pat. No. 4,761,406, or in combination withone or more bone therapeutic agents such as, without limitation,calcitonin and estrogen.

PTH Analog Receptor-Signaling Activities

A crucial step in the expression of hormonal action is the interactionof hormones with receptors on the plasma membrane surface of targetcells. The formation of hormone-receptor complexes allows thetransduction of extracellular signals into the cell to elicit a varietyof biological responses.

Polypeptides described herein can be screened for their agonistic orantagonistic properties using the cAMP accumulation assay. Cellsexpressing PTH-1 receptor on the cell surface are incubated with nativePTH(1-84) for 5-60 minutes at 37° C., in the presence of 2 mM IBMX(3-isobutyl-1-methyl-xanthine, Sigma, St. Louis, Mo.). Cyclic AMPaccumulation is measured by specific radio-immunoassay. A compound thatcompetes with native PTH(1-84) or PTH(1-34) for binding to the PTH-1receptor, and that inhibits the effect of native PTH(1-84) or PTH(1-34)on cAMP accumulation, is considered a competitive antagonist. Such acompound would be useful for treating hypercalcemia.

Conversely, a PTH analog described herein or a derivative thereof thatdoes not compete with native PTH(1-84) or PTH(1-34) for binding to thePTH-1receptor, but which still prevents native PTH(1-84) or PTH(1-34)activation of cAMP accumulation (presumably by blocking the receptoractivation site) is considered a non-competitive antagonist. Such acompound would be useful for treating hypercalcemia.

The compounds described herein that compete with native PTH(1-84) orPTH(1-34)) for binding to the PTH-1 receptor, and which stimulates cAMPaccumulation in the presence or absence of native PTH(1-84) or PTH(1-34)are competitive agonists. A compound that does not compete with nativePTH(1-84) or PTH(1-34) for binding to the PTH-1 receptor but which isstill capable of stimulating cAMP accumulation in the presence orabsence of native PTH(1-84) or PTH(1-34), or which stimulates a highercAMP accumulation than that observed by a compound of the invention or aderivative thereof alone, would be considered a non-competitive agonist.

Therapeutic Uses of PTH Analogs

Some forms of hypercalcemia and hypocalcemia are related to theinteraction between PTH and PTHrP and the PTH-1 and receptors.Hypercalcemia is a condition in which there is an abnormal elevation inserum calcium level; it is often associated with other diseases,including hyperparathyroidism, osteoporosis, carcinomas of the breast,lung and prostate, epidermoid cancers of the head and neck and of theesophagus, multiple myeloma, and hypernephroma. Hypocalcemia, acondition in which the serum calcium level is abnormally low, may resultfrom a deficiency of effective PTH, e.g., following thyroid surgery.

By “agonist” is intended a ligand capable of enhancing or potentiating acellular response mediated by the PTH-1 receptor. By “antagonist” isintended a ligand capable of inhibiting a cellular response mediated bythe PTH-1 receptor. Whether any candidate “agonist” or “antagonist” ofthe present invention can enhance or inhibit such a cellular responsecan be determined using art-known protein ligand/receptor cellularresponse or binding assays, including those described elsewhere in thisapplication.

In accordance with yet a further aspect of the invention, there isprovided a method for treating a medical disorder that results fromaltered or excessive action of the PTH-1 receptor, comprisingadministering to a patient therapeutically effective amount of acompound of the invention or a derivative thereof sufficient to inhibitactivation of the PTH-1 receptor of said patient.

In this embodiment, a patient who is suspected of having a disorderresulting from altered action of the PTH-1 receptor can be treated usingcompounds of the invention or derivatives thereof of the invention whichare a selective antagonists of the PTH-1 receptor. Such antagonistsinclude compounds of the invention or derivatives thereof of theinvention which have been determined (by the assays described herein) tointerfere with PTH-1 receptor-mediated cell activation or otherderivatives having similar properties.

To administer the antagonist, the appropriate compound of the inventionor a derivative thereof is used in the manufacture of a medicament,generally by being formulated in an appropriate carrier or excipientsuch as, e.g., physiological saline, and preferably administeredintravenously, intramuscularly, subcutaneously, orally, or intranasally,at a dosage that provides adequate inhibition of a compound of theinvention or a derivative thereof binding to the PTH-1 receptor. Typicaldosage would be 1 ng to 10 mg of the peptide per kg body weight per day.

In accordance with yet a further aspect of the invention, there isprovided a method for treating osteoporosis, comprising administering toa patient a therapeutically effective amount of a compound of theinvention or a derivative thereof, sufficient to activate the PTH-1receptor of said patient. Similar dosages and administration asdescribed above for the PTI/PTHrP antagonist, can be used foradministration of a PTH/PTHrP agonist, e.g., for treatment of conditionssuch as osteoporosis, other metabolic bone disorders, andhypoparathyroidism and related disorders.

It will be appreciated to those skilled in the art that the inventioncan be performed within a wide range of equivalent parameters ofcomposition, concentration, modes of administration, and conditionswithout departing from the spirit or scope of the invention or anyembodiment thereof.

Having now fully described the invention, the same will be more readilyunderstood by reference to specific examples which are provided by wayof illustration, and are not intended to be limiting of the invention,unless herein specified.

EXAMPLES

The following protocols and experimental details are referenced in theexamples that follow.

Peptides. Each peptide utilized in this study contained a free aminoacid terminus and a carboxamide at the C-terminus. Peptides wereprepared on automated peptide synthesizers (model 430A PE, AppliedBiosystems, Foster City, Calif., or Model 396 MBS Advanced Chem Tect,Louisville, Ky.) using Fmoc main-chain protecting group chemistry,HBTU/HOBt/DIEA (1:1:2 molar ratio) for coupling reactions, andTFA-mediated cleavage/sidechain-deprotection (MGH Biopolymer SynthesisFacility, Boston, Mass.). All peptides were desalted by adsorption on aC18-containing cartridge, and purified further by HPLC. The dry peptidepowders were reconstituted in 10 mM acetic acid and stored at −80° C.The purity, identity, and stock concentration for each peptide wassecured by analytical HPLC, Matrix-assisted laser desorption/ionization(MALDI) mass spectrometry and amino acid analysis. Radiolabeling of[M]PTH(1-21) and [Aib^(1,3),M]PTH(1-21) was performed using ¹²⁵I-Na(2,200 Ci/mmol, NEN) and chloramine-T; the resultant radioligands werepurified by HPLC.

Cell Culture. The cell line HKRK-B28 (Takasu, H., et al., J. Bone Miner.Res. 14:11-20 (1999)) was derived from the porcine kidney cell line,LLC-PK₁ by stable transfection with plasmid DNA encoding the human P1Rand expresses ˜280,000 receptors per cell. These cells, as well as COS-7cells and SaOS-2-B10 cells, were cultured at 27° C. in T-75 flasks (75mm²) in Dulbecco's modified Eagle's medium (DMEM) supplemented withfetal bovine serum (10%), penicillin G (20 units/ml), streptomycinsulfate (20 μg/ml) and amphotericin B (0.05 μg/ml) in a humidifiedatmosphere containing 5% CO₂. Stock solutions of EGTA/trypsin andantibiotics were from GIBCO; fetal bovine serum was from HycloneLaboratories (Logan, Utah). COS-7 cells sub-cultured in 24-well plateswere transfected with plasmid DNA (200 ng per well) encoding thewild-type human P1R or truncated human P1R deleted for residues (24-181)(Shimizu, M., et al., J. Biol. Chem. 275:21836-21843 (2000)) that waspurified by cesium chloride/ethidium bromide density gradientcentrifugation, and FuGENE 6 transfection reagent (Roche IndianapolisInd.) according to the manufacturer's recommended procedure. All cells,in 24-well plates, were treated with fresh media and shifted to 33° C.for 12 to 24 h prior to assay.

cAMP Stimulation. Stimulation of cells with peptide analogs wasperformed in 24-well plates. Cells were rinsed with 0.5 mL of bindingbuffer (50 mM Tris-HCI, 100 nM NaCI, 5 mM KCl, 2 mM CaCl₂, 5%heat-inactivated horse serum, 0.5% fetal bovine serum, adjusted to pH7.5 with HCl) and treated with 200 μL of cAMP assay buffer (Delbecco'smodified Eagle's medium containing 2 mM 3-isobutyl-1-methylxanthine, 1mg/mL bovine serum albumin, 35 mM Hepes-NaOH, pH 7.4) and 100 μL ofbinding buffer containing varying amounts of peptide analog (finalvolume=300 μL). The medium was removed after incubation for 30 to 60minutes at room temperature, and the cells were frozen on dry ice, lysedwith 0.5 mL 50 mM HCl, and refrozen (˜80° C). The cAMP content of thediluted lysate was determined by radioimmunoassay. The EC₅₀ responsevalues were calculated using nonlinear regression (see below).

Competition Binding. Binding reactions were performed with HKRK-B28cells or in COS-7 cells in 24-well plates. The cells were rinsed with0.5 mL of binding buffer, and then treated successively with 100 μLbinding buffer, 100 μL of binding buffer containing various amounts ofunlabeled competitor ligand, and 100 μL of binding buffer containing ca.100,000 cpm of ¹²⁵I-[M]PTH(1-21) or ¹²⁵I-[Aib^(1,3),M]PTH(1-21)} (ca. 26fmol; final volume=300 μL). Incubations were 4 to 6 h at 4° C., at whichtime near equilibrium conditions were attained. Cells were then placedon ice, the binding medium was removed, and the monolayer was rinsedthree times with 0.5 mL of cold binding buffer. The cells weresubsequently lysed with 0.5 mL 5N NaOH and counted for radioactivity.For each tracer and in each experiment, the non-specific binding wasdetermined as the radioactivity that bound in the presence of the sameunlabeled peptide at a concentration of 1 μM, and was ˜1% of totalradioactivity added for each tracer. The maximum specific binding (B₀)was the total radioactivity bound in the absence of competing ligand,corrected for nonspecific binding, and for each tracer, ranged from 8%to 20% of the total radioactivity added. Nonlinear regression was usedto calculate binding IC₅₀ values (see below). Scatchard transformationsof homologous competition binding data derived from studies with 26 fmolof ¹²⁵I-[Aib^(1,3),M]PTH(1-21) were employed for estimations of apparentequilibrium dissociation constant (k_(Dapp)s) and total number of ligandbinding sites (B_(max)), assuming a single class of binding sites andequal affinities of the iodinated and non iodinated ligand.

Stimulation of Inositol Phosphate Production. COS-7 cells transfected asabove with P1R-WT were treated with serum-free, inositol-free DMEMcontaining 0.1% bovine serum albumin and [³H]myo-inositol (NEN, Boston,Mass.) (2 μCi/mL) for 16 h prior to assay. At the time of the assay, thecells were rinsed with binding buffer containing LiCl (30 mM) andtreated with the same buffer with or without a PTH analog. The cellswere then incubated at 37° C. for 40 min, after which the buffer wasremoved and replaced by 0.5 mL of ice cold 5% trichloroacetic acidsolution. After 3 h on ice, the lysate was collected and extracted twicewith ethyl ether. The lysate was then applied to an ion exchange column(0.5 mL resin bed) and the total inositol phosphates were eluted asdescribed previously (Berridge, M. J., et al., Biochem. J. 212:473-482(1983)), and counted in liquid scintillation cocktail.

Inhibition of Chondrocyte Differentiation in Embryonic MouseMetatarsals. Metatarsals from embryonic day (E) 15.5 mouse embryos wereexcised and cultured in a 37° C. humidified incubator (5% CO₂) inserum-free αXMEM media in 24 well plates. Sixteen hours later, a PTHanalog or vehicle was added, and the samples were incubated for anadditional 48 h in 37° C. with peptide or vehicle added again at the 24h time point. At the end of the 64 h incubation period, the samples werefixed with 10% formalin/phosphate-buffered saline, then directlyvisualized on a dissecting microscope using white light. Sections wereprocessed for in-situ hybridization analysis using ³⁵S-labeledriboprobes specific for collagen type X mRNA, a developmental markergene expressed only in hypertrophic chrondrocytes of the growth plate.

Circular Dichroism. Circular Dichroism spectra were recorded on a Jascomodel 710 spectropolarimeter; peptides were analyzed at a concentrationof20 μM in 50 mM sodium phosphate buffer pH 7.4, or the same buffercontaining 2,2,2-trifluoroethanol at 20% (v/v). Spectroscopic scans werepreformed at 20° C. and at wavelengths between 185 and 255 nM, with datarecored at each 1 nM interval. The spectral bandwidth was 1.5 nM and 8scans were accumulated and averaged for each sample. At each wavelength,the mean residue elipticity [θ×100/l×C×n); where θ is the raw elipticityvalue (in dimensions of millidegree), l is the sample path length, C=isthe molar peptide concentration, and n is the number of residues in thepeptide (Bowen, W. P., and Jerman, J. C., Trends in Pharmacol. Sci. 16:413-417 (1995)). The helical content of each peptide was estimated bydividing [θ] observed at 222 nM for that peptide by −28,100, which isthe reported [θ]₂₂₂obs for a model helical decapeptide (Bowen, W. P.,and Jerman, J. C., Trends in Pharmacol. Sci. 16: 413-417 (1995)).

Data Calculation. Calculations were performed using Microsoft® Excel.Nonlinear regression analyses of binding and cAMP dose-response datawere performed using the four-parameter equation:y_(p)=Min+[(Max−Min)/(1+(IC₅₀/x)^(slope))]. The Excel Solver functionwas utilized for parameter optimization, as described previously(Carter, P. H., et al., Endocrinology 140: 4972-4981 (1999); Bowen, W.P., and Jerman, J. C., Trends in Pharmacol. Sci. 16: 413-417 (1995)).Differences between paired data sets were statistically evaluated usinga one-tailed Student's t-test, assuming unequal variances for the twosets.

Example 1 Aib-scan in [M]PTH(1-14)

The effect of introducing individual Aib substitutions at each positionin the scaffold peptide [M]PTH(1-14) (Shimizu, M., et al., Endocrinology(2001) (In Press))) were analyzed. In cAMP stimulation assays inHKRK-B28 cells, the parent peptide [M]PTH(1-14) stimulated approximatelythe same (˜70-fold) maximun (Emax) increase in intracellular cAMP thatwas induced by PTH(1-34), but the potency (EC₅₀) of the shorter peptidewas 40-fold less than that of PTH(1-34) (EC₅₀ s=100±20 and 2.5±0.4. nM,respectively) (FIG. 1 and Table 1). Most of the Aib substitutionsdiminished potency. Severe reductions occured with Aib substitutions atpositions 6, 8 and 9 (all>2,300-fold), moderate reductions occurred withsubstitution at positions 2, 4, 5 and 11 (all 170 to 670-fold) and minorreductions occurred with substitutions at positions 7, 10, 12, 13 and 14(all<3-fold; Table 1). Substitution of Aib at positions 1 and 3 resultedin peptides with 10- and 8-fold improvements in potency, relative to[M]PTH(1-14), respectively (P≦0.01). These Aib-scan data extend previousalanine-scan and substitution analyses of PTH(1-14) analogs, in whichresidues in the (1-9) region, excluding residue 3, were found to beintolerant to substitution, and residues 3 and 10-14. were found to berelatively tolerant (Luck, M. D., et al., Molecular Endocrinology13:670-680 (1999); Shimizu, M., et al., J. Biol. Chem. 275:19456-19460(2000); Pellegrini, M., et al., J. Biol. Chem. 273:10420-10427 (1998)).

The P1R-binding properties of these analogs were assayed in competitionstudies performed in HKRK-B8 cells. In previous studies, PTH(1-14)binding could not be detected using ¹²⁵I-PTH(1-34) and relatedN-terminally intact and relatively unmodified radioligands (Luck, M. D.,et al., Molecular Endocrinology 13:670-680 (1999)). However, measurablePTH(1-14) binding was observed with ¹²⁵I-PTH(3-34) used as a tracerradioligand (Hoare, S. R. J., et al., J. Biol. Chem 276:7741-7753(2001); Shimizu, M., et al., Endocrinology (2001) (In Press)). Receptorbinding affinity was assessed using a tracer radioligand that wasstructurally more homologous to the [M]PTH(1-14) analogs beinginvestigated. The radiolabeled peptide¹²⁵I-[Ala^(3,12)Nle⁸Gln¹⁰,Har¹¹,Trp¹⁴,Tyr¹⁵]PTH(1-15)amide wasevaluated, but did not bind detectably to HKRK-B28 cells. A similaranalog, which was extended to position 21 and contained theaffinity-enhancing substitution of Glu¹⁹→Arg (Takasu, H., et al.,Biochemistry 38:13453-13460 (1999); Kronenberg, H. M., et al., RecentProg. Horm. Res. 53:283-301 (1998)), was prepared. The resultingradioligand ¹²⁵I-[M]PTH(1-21) bound adequately to the P1R expressedintact HKRK-B28 cells, as the amount of specifically bound radioactivity(e.g. that which could be inhibited by excess unlabeled [M]PTH(1-2)peptide), was ˜15% to 20% of total radioactivity added, and that whichbound to untransfected LLC-PK1 cells was<2% of total added. Thus, thistracer ligand was suitable for competition analyses.

The binding of ¹²⁵I-[M]PTH(1-21) to HKRK-B28 cells was fully inhibitedby PTH(1-34) (IC₅₀=18±3 nM) and more weakly but to near completion by[M]PTH(1-14) (IC₅₀=13,000±3,000 nM, Table 1). Relative to the apparentbinding affinity of [M]PTH(1-14), most of the Aib substitutions reducedaffinity, in accordance with the corresponding effects on cAMP-signalingpotency (Table 1). The only Aib substitutions that improved affinitysignificantly were those at positions 1 and 3 (13- and 8-fold,respectively, Aib at position 10 showed a trend towards causing a1.4-fold improvement in affinity, P=0.16). Strong (>10-fold) reductionsin affinity occurred with Aib substitutions at positions 4, 7, 8 and 9,while mild (<10-fold) reductions occurred with the Aib at positions 2,5, 12, 13 and 14. While most of the Aib substitutions had effects onreceptor-binding affinity that were approximately proportional to theireffects on cAMP-stimulating potency, those at positions 2 and 6 had lessof an effect on binding than on potency. Thus, these two substitutionsreduced affinity ˜3-fold, relative to [M]PTH(1-14), while they reducedpotency ˜470- and ˜2,300-fold, respectively (Table 1).

Combining the Aib substitutions at positions 1 and 3 revealed anadditive effects, as [Aib^(1,3),M]PTH(1-14) was 90-fold more potent instimulating cAMP formation than was [M]PTH(1-14) (EC₅₀ s=1.1±0.1 nM,100±20 nM, respectively), and at least as potent as PTH(1-34)(EC₅₀=2.5±0.4 nM, P=0.01, FIG. 2A and Tale 1). The effects of the singleAib substitutions at position 1 and 3 on receptor-binding affinity werealso additive, as [Aib^(1,3),M]PTH(1-14) bound with 100-fold higherapparent affinity than did [M]PTH(1-14) (FIG. 2B and Table 1). Aibsubstitutions were subsequently introduced at positions 1 and 3 in[M]PTH(1-11) analog to determine if the paired substitution couldenhance activity of the shorter peptide sequence. Previously, it wasshown that while native PTH peptides shorter than (1-14) were devoid ofcAMP-stimulating activity (Luck, M. D., et al., Molecular Endocrinology13:670-680 (1999)), modified PTH(1-11) analogs, such as [M]PTH(1-11)could induce a full cAMP response in HKRK-B28 cells, albeit with apotency (EC₅₀=3 μM) nearly 1,000-fold weaker than that ofPTH(1-34)(Shimizu, M., et al., Endocrinology (2001) (In Press)). In cAMPstimulation assays in HKRK-B28 cells, [Aib^(1,3),M]PTH(1-11) was fullyefficacious and its potency (EC₅₀ 4.0±0.8 nM) was 1,000-fold greaterthan that of [M]PTH(1-11) (Shimizu, M., et al., Endocrinology (2001) (InPress)) and nearly equal to that of PTH(1-34) (FIG. 2A, Table 1). TheAib-1,3 modification also enhanced potency of PTH(1-10) analog, as[Aib^(1,3),Gln¹⁰]PTH(1-10) was 50-fold more potent than our previouslymost potent PTH(1-10) analog, [Ala³,Gln¹⁰]PTH(1-10) (EC₅₀ s=16±2 μM and˜800 μM, respectively) (Shimizu, M., et al., Endocrinology (2001) (InPress)) (FIG. 2A, Table 1). The 4000-fold weaker potency that[Aib^(1,3),Gln¹⁰]PTH(1-10) exhibited, relative to that of[Aib^(1,3),M]PTH(1-1 1), indicated the importance of the position 11residue (homoarginine) in the activities of the Aib-containing peptides. Little or no stimulation of cAMP accumulation was observed with[Aib^(1,3)]PTH(1-9) (FIG. 2A and Table 1). In competition bindingassays, [Aib^(1,3),M]PTH(1-11) effectively inhibited¹²⁵I-[Aib^(1,3),M[PTH(1-21) binding to HKRK-B8 cells (IC₅₀=970±300 nM),but [Aib^(1,3),Gln¹⁰]PTH(1-10) and [Aib^(1,3)]PTH(1-9) did not binddetachably (FIG. 2B and Table 1). TABLE 1 cAMP Stimulation andhP1R-Binding Properties in HKRK-B28 cells cAMP EMax_((obs.,)) BindingEC₅₀ pmole/ IC₅₀ # Peptide nM well (n) nM (n)  92 PTH(1-34) 2.5 ± 0.4280 ± 11 10 18 ± 3  7 621 [M]PTH(1-14) 100 ± 20  270 ± 8  10 13,000 ±3,000  4 Aib scan in [M]PTH(1-14) 622 Aib-1 10 ± 3  273 ± 6  6 980 ± 1603 623 Aib-2 47,000 ± 13,000 168 ± 6  3 50,000 ± 11,000 3 624 Aib-3 13 ±3  269 ± 7  6 1,700 ± 200   3 625 Aib-4 17,000 ± 3,400  221 ± 10 3148,000 ± 40,000  3 626 Aib-5 66,000 ± 38,000 169 ± 18 3 N.B. 3 627Aib-6 230,000 ± 78,000  116 ± 12 3 31,000 ± 7,000  3 628 Aib-7 2,600 ±980   275 ± 8  3 490,000 ± 170,000 3 629 Aib-8 1,500,000 ± 970,000   34± 7 3 N.B. 3 630 Aib-9 710,000 ± 330,000 51 ± 8 4 N.B. 3 631 Aib-103,000 ± 2,100 214 ± 22 3 9,100 ± 1,500 3 632 Aib-11 67,000 ± 51,000  96± 18 4 N.B. 3 633 Aib-12 440 ± 300 263 ± 9  3 15,000 ± 3,000  3 634Aib-13 480 ± 250 259 ± 10 4 44,000 ± 8,000  3 635 Aib-14 350 ± 100 273 ±6  3 79,000 ± 27,000 3 Aib-1,3 in [M]PTH(1-X) 608 [M]PTH(1-21) N.D. 53 ±4  3 674 [Aib^(1,3), M]PTH (1-21) 4.3 ± 1.6 284 ± 76 3 31 ± 7  3 671[Aib^(1,3), M]PTH (1-14) 1.1 ± 0.1 278 ± 20 5 130 ± 20  5 682[Aib^(1,3), M]PTH (1-11) 4.0 ± 0.8 243 ± 15 4 970 ± 300 3 684[Aib^(1,3), M]PTH (1-10) 16,000 ± 2,000  111 ± 8  4 N.B. 3 696[Aib^(1,3)]PTH (1-9) >10,000 10 ± 1 3 N.B. 3Peptides PTH(1-34) ([Nle^(8,21), Tyr³⁴]PTH(1-34)amide), [M]PTH(1-14) (M= Ala^(3,12), Gln¹⁰, Har¹¹, Trp¹⁴), and [M]PTH(1-14 analogs, orC-terminally truncated derivatives thereof, containing α-aminoisobutyricacid (Aib) substitutions at the indicated positions were functionallyevaluated in HKRK-B28 cells.“M” in [Aib^(1,3), M]PTH(1-21) = Nle⁸, Gln¹⁰, Har¹¹, Ala¹², Trp¹⁴, Arg¹⁹and Tyr²¹.The basal cAMP values (not subtracted) were 4.0 ± 0.1 pmole/well (n =10). Peptides were based on the rat PTH sequence and werecarboxy-amidated. Competition binding analyses were performed with¹²⁵I-[M]PTH(1-21 )amide (Ala^(1,3)) as tracer for 4 h at 4° C. Data aremeans (±S.E.M.) Of the number of experiments indiated (n).N.B., no binding was detected at a peptide concentration of 10 μm; N.D.;the experiment was not done.The sequence of MPTH(1-14) is:Ala-Val-Ala*-Glu-Ile-Gln-Leu-Met-His-Gln*-Har*-Ala*-Lys-Trp* where theasterisk denotes substituted amino acids are not found at that positionin native rat PTH(1-14).

Example 2 Analog Activity in COS-7 Cells

The possibility that the activity-enhancing effects of the Aibsubstitutions at positions 1 and 3 were mediated through thejuxtamembrane (J) region of the receptor was investigated in COS-7 cellstransiently transfected with P1R-delNt. P1R-delNt was a truncated P1Rthat lacked most of the amino-terminal extracellular domain. With thisreceptor construct, PTH(1-34) was a much weaker agonist than it was withP1R-WT, while other PTH(1-14) analogs exhibited approximately the samepotency with P1R-delNt as with P1R-WT (Kaul, R., and Balaram, P.,Bioorganic & Medicinal Chemistry 7:105-117 (1999)). Consistent withthis, the cAMP-stimulating potency of [Aib^(1,3),M]PTH(1-14) onP1R-delNt (EC₅₀=0.73±0.16 nM) was comparable to its potency on COS-7cells expressing P1R-WT (1.2±0.6 nM) (Table 2). On P1R-delNt,[Aib^(1,3),M]PTH(1-14) was 55-fold more potent than was [M]PTH(1-14)(EC₅₀=40±2 nM, FIG. 3A and Table 2). This result indicated that thepotency-enhancing effects of the Aib-1,3 substitutions were exertedthrough the J domain of the receptor. Remarkably, [Aib^(1,3),M]PTH(1-14)was as potent on P1R-delNt as PTH(1-34) was on P1R-WT (EC₅₀s=0.73±0.16nM and 1.4±0.7 nM, respectively, P=0.4, Table 2) and the EMax induced by[Aib^(1,3),M]PTH(1-14) on P1R-delNt was equal to that induced byPTH(1-34) on P1R-WT (250±20 picomole/well and 240±50 picomole/well,respectively, P=0.7, Table 2). As expected, PTH(1-34), was ˜500-foldweaker on P1R-delNt than P1R-WT (EC₅₀s=680±110 nM and 1.4±0.7 nM,respectively; FIG. 3A and Table 2).

Heretofore, it was not possible to demonstrate a PLC response for anyPTH analog in cells expressing P1R-delNt, including [M]PTH(1-14). Theanalog [Aib^(1,33),M]PTH(1-14), however, induced an approximate 3-foldincrease in inositol phosphate (IP) production, relative to the basallevel of IPs, in COS-7 cells expressing P1R-delNt, while, as expected,PTH(1-34) and [M]PTH(1-14) were inactive (FIG. 3B). Thus, the truncatedreceptor can couple to the PCL signaling pathway when stimulated withthe Aib-containing PTH peptide. With P1R-WT, both [Aib^(1,3),M]PTH(1-14)and [M]PTH(1-14) stimulated the same 4-fold increase in IP formationthat was observed for PTH(1-34) acting on this receptor, and with thisreceptor, [Aib^(1,3),M]PTH(1-14) was 66-fold more potent than[M]PTH(1-14) (EC₅₀=71±9 nM, and 4,700±2,000 nM, respectively, Table 2).Thus, the Aib-1,3 substitutions enhance the ligand's capacity tostimulate PLC activity with P1R-WT, as well as with P1R-delNt.

The radioligand used in the above binding studies HKRK-B8 cells,¹²⁵I-[M]PTH(1-21), did not bind detectably to P1R-delNt. To potentiallyimprove affinity of this peptide, the paired Aib-1,3 modifications wereintroduced to produce [Aib^(1,3),M]PTH(1-21). The correspondingradioiodinated peptide, ¹²⁵I-[Aib^(1,3),M]PTH(1-21), bound to COS-7cells expressing P1R-WT; thus, the specific binding of this tracer (e.g.that which could be inhibited by excess unlabeled [Aib^(1,3),M]PTH(1-21)peptide) was ˜10% and ˜15% of the total radioactivity added, for eachreceptor, respectively. The total specific binding observed in COS-7cells tranfected with vector DNA alone was<2% of total radioactivityadded. This radioligand, therefore, enabled competition bindingexperiments to be performed with both the wild-type and truncated PTH-1receptors. Scatchard transformation of homologous competition bindingdata obtained with ¹²⁵I-[Aib^(1,3), M]PTH(1-21) as tracer radioligandand varying amounts of unlabeled [Aib^(1,3),M]PTH(1-21) indicated thatthe ligand's affinity at P1R-delNt was slightly (<2-fold) weaker than itwas at P1R-WT (K_(Dapp)s=29±3 and 17±2 nM, respectively, P=0.01), whilethe corresponding B_(max) values for the two receptors were notsignificantly different (1.3±0.1×10⁶ receptors/cell and 1.9±0.8×10⁶receptors/cell respectively, P=0.3).

At the truncated receptor, [Aib^(1,3),M]PTH(1-14), in addition to[Aib^(1,3),M]PTH(1-21), effectively inhibited the binding of¹²⁵I-[Aib^(1,3),M]PTH(1-21), whereas, PTH(1-34) did not (FIG. 3C). Like[Aib^(1,3),M]PTH(1-21), the apparent binding affinities that[Aib^(1,3),M]PTH(1-14) and [M]PTH(1-14) exhibited at P1R-delNt werecomparable to the corresponding affinities observed at the wild-type P1R(Table 2). At both P1R-delNt and P1R-WT, the binding affinities of[Aib^(1,3),M]PTH(1-14)were ˜10-fold stronger than the correspondingaffinities observed for [M]PTH(1-14). The Aib substitutions thereforeenhanced the ligand's binding affinity for the J domain of the P1R.TABLE 2 Functional properties of PTH analogs in COS-7 cells cAMP PLCBinding EC₅₀ EMAX_((obs.)) EC₅₀ EMAX_((obs.)) IC₅₀ Peptide^(a) nMpmole/well (n) nM foldxbasal (n) nM (n) hP1R-WT 92.5 PTH(1-34) 1.4 ± 0.7240 ± 50 3 17 ± 2  4.2 ± 0.6 3 12 ± 3  4 521 PTH(1-14) 90,000 ± 34,000 140 ± 30* 3 — N.B. 3 621 [M]PTH(1-14) 49 ± 21 240 ± 50 3 4,700 ± 2,0003.9 ± 0.5 3 20,000 ± 3,000  3 674 [Aib^(1,3)M]PTH(1-21) 0.8 ± 0.1 300 ±30 3 N.D. 28 ± 5  6 671 [Aib^(1,3)M]PTH(1-14) 1.2 ± 0.6 240 ± 40 3 71 ±9  4.3 ± 0.6 3 2,250 ± 1,100 4 682 [Aib^(1,3)M]PTH(1-11) 2.1 ± 0.7 190 ±40 3 N.D. 16,000 ± 1,000  3 684 [Aib^(1,3)M]PTH(1-10) 100,000 ± 40,000  120 ± 10* 3 N.D. N.B. 3 hP1R-delNT 92.5 PTH(1-34) 680 ± 110 220 ± 30 3— 3 N.B. 4 521 PTH(1-14) 140,000 ± 30,000   110 ± 10* 3 — N.B. 3 621[M]PTH(1-14)  40 ± 2.0 220 ± 20 3 — 3 17,400 ± 1,400  3 674[Aib^(1,3)M]PTH(1-21) 0.38 ± 0.10 240 ± 20 3 N.D. 27 ± 1  6 671[Aib^(1,3)M]PTH(1-14) 0.73 ± 0.16 250 ± 20 3 130 ± 30  3.1 ± 0.2 3 1,600± 200   4 682 [Aib^(1,3)M]PTH(1-11) 2.00 ± 0.40 220 ± 20 3 N.D. 13,000 ±1,000  3 684 [Aib^(1,3)M]PTH(1-10) 53,000 ± 10,000  84 ± 4* 3 N.D. N.B.3The peptides were derivatives of rat PTH with C-terminal carboxamides;in PTH(1-14) and shorter analogs, “M” refers to the amino acidmodifications: Ala^(3,12.) Gln¹⁰, Har¹¹, Trp¹⁴, unless the residueposition was absent due to truncation or replaced by Aib(α-aminoisobutyric acid); in the PTH(1-21) analog, “M” refers to thesame modifications and the modifications# of Nle⁸, Arg¹⁹ and Tyr²¹. Peptides were evaluated in COS-7 cellstransiently transected with either the wild-type hP1R (hP1R-WT), or atruncated hP1R lacking most of the amino-terminal extracellular domain(hP1R-delNt).The basal levels of cAMP were 10.3 ± 1.1 and 9.5 ± 1.3 picomole per wellfor hP1R-WT and hP1R-delNt, respectively.The basal levels of ³H-inositol phosphates were 1,103 ± 143 and 2,929 ±877 cpm per well for hP1R-WT and hP1R-delNt, respectively.TheEmax^((obs.) (maximum response observed) values in the cAMP and PLC assays were determined at ligand doses of 0.1 to 100 μM; an asterisk indicated that a plateau in the response curve was not attained and the curve-fitting equation used to determine the EC)₅₀ was constrained to within one standard deviation of the maximumresponse observed with the same receptor in the same assay. Competitionbinding assays were performed# with ¹²⁵1-[Aib^(1,3), M]PTH(1-21) radioligand as tracer.Values are means (±S.E.M.) of data from the number of independentexperiments indicated (n), each of which was performed in duplicate.A dashed line indicates that no cAMP or PLC response was observed.N.B. indicates that no inhibition of tracer binding was observed.N.D. indicates that the experiment was not done.

Example 3 Activity in Bone Cells

The number of PTH-1 receptors expressed on the surface of the PTH targetcells in bone or kidney is uncertain, but it is likely to beconsiderably lower than that found in HKRK-B28 cells. Therefore, severalof the Aib-mounted PTH analogs were evaluated using SaOS-2 cells. Thesecells were derived from a human osteosarcoma, exhibited osteoblast-likeproperties and endogenously expressed relatively low levels of the PTH-1receptor (˜20,000 receptors/cell (Marx, U. C., et al., J. Biol. Chem.273:4308-4316 (1998)). In these cells, [Aib¹,M]PTH(1-14) and[Aib³,M]PTH(1-14) were 15- and 8-fold more potent in stimulating cAMPformation than was [M]PTH(1-14), and [Aib^(1,3),M]PTH(1-14) was 130-foldmore potent than [M]PTH(1-14) (FIG. 4 and Table 3). Thus, in SaOS-2cells, [Aib^(1,3),M]PTH(1-14) was only 13-fold less potent thanPTH(1-34) and at least five-orders of magnitude more potent than nativePTH(1-14), for which no activity could be detected, even at a dose of 10μM (FIG. 4).

Whether or not [Aib^(1,3),M]PTH(1-14) activity could be detected in amore intact bone system was studied in an explant assay. An explantassay utilized cartilaginous metatarsal rudiments isolated fromE15.5mouse embryos and subsequently cultured in multi-well platescontaining serum-free media. A PTH peptide analog or vehicle control,was added to the culture 16 h after explantion, then again at 24 h. Theincubation was terminated 24 h later for a total of 48 h of treatmentover a 64 h period. In the absence of PTH, chondrocyte differentationoccurred, such that by the end of the experiment, dense mineralizationwas apparent at the bone's mid-section (FIG. 5A). Differentiation wasinhibited by the presence of PTH(1-34) (0.1 μM) or[Aib^(1,3),M]PTH(1-14) (1μM), as no mineralization was observed (FIGS.5, B and C). Mineralization was also inhibited in these assays by[Aib^(1,3),M]PTH(1-14), whereas no effect could be detected for nativePTH(1-14) (2 μM) (FIG. 5D). Comparable results were obtained in each ofthree replicate experiments. In addition, mRNA in situ hybridizationanalysis performed on the explanted metatarsals demonstrated that bothPTH(1-34) and [Aib^(1,3),M]PTH(1-14) inhibited expression of thecollagen X gene, a bone developmental marker gene (data not shown).These inhibitory effects were consistent with the known capacity ofPTHRP to retard chondrocyte differentiation in the growth platecartilage of developing long bones (Pellegrini, M., et al., Biochemistry37:12737-12743 (1998)). TABLE 3 cAMP Stimulation in SaOS-2 cells PeptideEC₅₀ nM EMAX_((obs.)) pmole/well n  93 PTH (1-34)  0.2 ± 0.02 350 ± 30 4621 [M]PTH(1-14) 340 ± 120 340 ± 30 4 521 PTH(1-14) N.R. 2 622 [Aib¹,M]PTH(1-14) 22 ± 4  340 ± 30 4 624 [Aib³, M]PTH(1-14) 42 ± 8  330 ± 30 4671 [Aib^(1,3), M]PTH(1-14) 2.6 ± 0.5 320 ± 30 3The peptides PTH(1-34) ([Nle^(8,21), Tyr³⁴]PTH(1-34)amide), [M]PTH(1-14)(m = Ala^(3,12), Gln¹⁰, Har¹¹, Trp¹⁴), native PTH(1-14), and analogsof[M]PTH(1-14) containing α-aminoisobutyric acid (Aib) at positions 1and/or 3, were evaluated for the capacity to stimulate cAMP productionin the human osteoblastic cell line SaOS-2.The calculated EC50 values and observed maximum response values aremeans (±S.E.M.) of data from the number of experiments indicated (n).The basal cAMP level was 6.4 ± 0.8 (n = 4).N.R. indicates that no cAMP response was detected.

Example 4 Circular Dichroism

Circular dichroism (CD) spectroscopy was used to analyze the potentialeffects that the Aib substitutions had on peptide secondary structurewhen the peptides were free in solution. Samples were analyzed in bothaqueous phosphate buffer and in phosphate buffer containing2,2,2-trifloroethanol, an organic solvent which promotes helicalstructure in oligopeptides, including PTH peptide fragments (Pellegrini,M., et al., J. Biol. Chem. 273:10420-10427 (1998); Gronwald, W., et al.,Biol. Chem. Hoppe Seyler 377:175-186 (1996); Barden, J. A., and Kemp, B.E., Biochemistry 32:7126-7132 (1993)). In phosphate buffer, the helicalcontent of each peptide, estimated from the elipticity observed at 222nM, was small (≦16%); however, [Aib^(1,3),M]PTH(1-14) contained nearlytwice as much helix as did [M]PTH(1-14) (16% and 8.1% respectively), asdid [Aib^(1,3),M]PTH(1-1l), as compared to [M]PTH(1-1) (13% and 7.5%respectively, Table 4). In 2,2,2-trifluoroethanol, the helical contentof each peptide increased; [Aib^(1,3),M]PTH(1-14) and[Aib^(1,3),M]PTH(1-11) exhibited the two highest levels of helicalcontent (56% and 57%, respectively) and were each more helical thantheir Ala-1,-3-containing counterpart peptides (FIG. 6 and Table 4). Thehigher helical contents of these two peptides were evident not only fromthe negative elipticities at 192 nM and 222 nM, but also from thepositive elipticities at 192 nM (FIG. 6). Unmodified PTH(1-11) exhibitedthe least amount of helical structure (30%), whereas[Aib^(1,3),M]PTH(1-10) was approximately 47% helical (FIG. 6 and Table4). These results suggest that the Aib-1,3 modifications increase thehelical structure of the N-terminal PTH oligo peptides in the freesolution phase. TABLE 4 Helicity in N-terminal PTH peptides [0]₂₂₂obs ×10⁻³ helical residues (%) peptide Phos. Phos + TFE Phos. Phos + TFEPTH(1-14) −2.6 −10.6 9.1 38 [M]PTH(1-14) −2.3 −11.9 8.1 42 Aib^(1,3),M]PTH(1-14) −4.6 −15.7 16 56 PTH(1-11) −1.8 −8.4 6.5 30 [M]PTH(-11) −2.1−9.9 7.5 35 [Aib^(1,3), M]PTH(1-11) −3.7 −16.1 13 57 [Aib^(1,3),M]PTH(1-10) −3.2 −13.1 11 47Circular dichroism spectra were recorded in either 50 mM phosphatebuffer or 50 mM phosphate buffer containing trifluoroethanol (20%) asdescribed in Material and Methods and shown in FIG. 6.The mean residue elipticity ([0]₂₂₂ ^(obs/)[0]₂₂₂ ^(max)) × 100; where[0]₂₂₂ obs is the mean residue elipticity at 222 nM observed for thatpeptide and [0]₂₂₂ max is the mean residue elipticity# reported for a model helical peptide of 10 amino acids (−28.1 × 10⁻³;Yang et al. 1986 Methods in Enzymol. 130, 208-269).

Example 5 PTH Analogs

As the first step, Aib was introduced at each position in [M]PTH(1-14).The Aib-scanning data indicated that substitutions at most positionsdiminished activity. However, the Aib scan data revealed considerable(8- to 10-fold) improvements in cAMP signaling potency withsubstitutions at position one and three, and these effects wereadditive, as [Aib^(1,3),M]PTH(1-14), with an EC₅₀ of ˜1 nM in HKRK-B28cells, was 100-fold more potent than [M]PTH(1-14), and at least aspotent as PTH(1-34).

Competition binding studies performed with ¹²⁵I-[M]PTH(1-21) indicatedthat most of the Aib substitutions exerted their effects on potency(positive or negative), at least in part, by changing PTH-1receptor-binding affinity. Thus, the Aib-1 and Aib-3 substitutions eachimproved the apparent affinity of [M]PTH(1-14) for HKRK-B28 cells byapproximately 10-fold, and the combined Aib-1,3 substitution increasedaffinity by approximately 100-fold. Likewise, the decreases in cAMPsignaling potency caused by most of the other Aib substitutions could beexplained by decreases in apparent binding affinity, even though,overall, binding affinities were generally 10- to 100-fold weaker thanthe corresponding cAMP signaling potencies. Two exceptions to this werethe peptides substituted at positions 2 and 6, at which signalingpotency was comparable with (position 2) or ˜10-fold weaker than thecorresponding apparent binding affinity. That the substitution of Aibfor valine-2 or glutamine-6 impaired signaling activity more thanreceptor-binding affinity, is consistent with the disproportionatereductions in signaling potency, relative to binding affinity, thatoccur with substitutions at these positions in PTH(1-34) analogs, and,in fact, result in PTH-1R antagonists (Cohen, F. E., et al., J. Biol.Chem. 266:1997-2004 (1991); Gardella, T. J., et al., J. Biol. Chem.266:13141-13146(1991), Carter 1999 #1180).

The 100-fold increase in cAMP-stimulating potency effect that occurredwith the paired Aib-1,3 modification to [M]PTH(1-14) seems consistentwith the hypothesis that an α-helix in the N-terminal portion of PTH isrequired for activation of the PTH-1 receptor. The capacity of Aib tostabilize α-helical structure in oligopeptides arises from the stericrestrictions on the rotations about the N-C° (φ) and C°-CO (ψ) bonds ofthe Aib residue that are imposed by the two methyl groups symmetricallybonded to its C° atom (Kaul, R., and Balaram, P., Bioorganic & MedicinalChemistry 7:105-117 (1999); Burgess, A. W., and Leach, S. J.,Biopolymers 12:2599-2605 (1973)). The φ and ψ torsion angles about thisC° atom are tightly restricted to those that occur in α-helices but thesymmetry of the di-allyl-substituted C° atom of Aib allows for eitherright-handed or left-handed α-helices. If the latter “reversed”configuration occurs in an otherwise right-handed helix, then the Aibresidue will, in all probability, induce a turn, and thus terminate thehelix (Kaul, R., and Balaram, P., Bioorganic & Medicinal Chemistry7:105-117 (1999); Venkataram Prasad, B. V., et al., Biopolymers18:1635-1646 (1979)). This reversed configuration is rare in peptidestructures, relative to the right-handed configuration (Kaul, R., andBalaram, P., Bioorganic & Medicinal Chemistry 7:105-117 (1999)), but itnevertheless leaves open the possibility that Aib at the amino-terminusof PTH(1-14) enhances potency through some mechanism other thanstabilization of an α-helix.

It is also of interest that the most beneficial effects on peptidepotency/affinity occurred with Aib substitutions at positions 1 and 3,since none of the structural studies on PTH(1-34) analogs have detectedstructure N-terminal of residue 3. It may be that Aib at position 1nucleates helix formation of “down-stream” residues within itselfparticipating in the helix. Alternatively, the modification may induceor stabilize helical structure at the very N-terminus of the peptidewhich is simply too unstable in the native sequence to be detected byNMR spectroscopy or x-ray crystallography. In any case, the 1000-foldhigher cAMP signaling potency exhibited by[Aib^(1,3),Gln¹⁰,Har¹¹]PTH(1-11) as compared to[Ala³,Gln¹⁰,Har¹¹]PTH(1-11) (EC₅₀s˜6 nM Vs. 3 μM, respectively, Table 1and (Shimizu, M., et al., Endocrinology (2001) (In Press)) demonstratesthat the effects of the Aib substitutions are exerted locally, e.g.within the first 11 amino acids of the peptide.

Direct structural analyses of these analogs, as free peptides, orpotentially in complex with the PTH-1 receptor, could provide valuableinsights into the ligand structures that allow a ligand to act as anagonist on the PTH-1 receptor. In this regard, the information derivedfrom the data set described herein could be of use in the design ofpeptide mimetics for the PTH-1 receptor. Approaching this problem fromthe standpoint of the native PTH peptide sequence is made difficult bythe conformational diversity that is possible at each position in thepeptide backbone chain. The incorporation of stereochemicallyconstrained amino acids, such as Aib, into the peptide chain, lessensthis problem, as it serves to nucleate predictable peptide structures.Thus, the approach can facilitate the de novo design of peptide ornonpeptide agonists for the PTH-1 receptor. Given the recently provenutility of PTH(1-34) in treating osteoporosis (Neer, R. M., et al.,N.E.J.M. 344:1434-1441 (2001)), such agonists should have importantmedical impact.

At the molecular level, it is presently unclear how the [Aib^(1,3),M]PTHanalogs interact with the receptor; nor is this known for any PTHligand, although fairly specific computer models of the interaction withnative PTH are now being developed (Jin, L., et al., J. Biol. Chem.275:27238-27244 (2000); Rölz, C., and Mierke, D. F., BiophysicalChemistry (2000) (In Press)). The above described experiments with thetruncated PTH-1 receptor, P1R-delNt, provide some insights, as theydemonstrate that the enhancing effects of the Aib substitutions atpositions 1 and 3 are mediated through the juxtamembrane region (Jdomain) of the receptor containing the extracellular loops andtransmembrane domains. This finding is consistent with the cumulativecrosslinking and mutational data on the PTH/PTH-1 receptor interaction,which indicate that residues in the (1-14) domain of PTH interactprimarily, if not exclusively, with the receptor's J domain, as opposedto its amino-terminal extracellular domain (N domain) (Bergwitz, C., etal., J. Biol. Chem. 271:26469-26472 (1996); Hoare, S. R. J., et al., J.Biol. Chem 276:7741-7753 (2001); Behar, V., et al., J. Biol. Chem.275:9-17 (1999); Shimizu, M., et al., J. Biol. Chem. 275:19456-19460(2000); Luck, M. D., et al., Molecular Endocrinology 13:670-680 (1999);Shimizu, M., et al., J. Biol. Chem. 275:21836-21843 (2000); Carter, P.H., and Gardella, T. J., Biochim. Biophys. Acta 1538:290-304 (2001);Gardella, T. J., et al., Endocrinology 132:2024-2030 (1993); Bisello,A., et al., J. Biol. Chem. 273:22498-22505 (1998)).

Another important conclusion to derive from our study with P1R-delNt, inwhich [Aib^(1,3),M]PTH(1-14) exhibited low nanomolar potency and fullefficacy in cAMP assays and nearly fill efficacy in PLC assays, is thatthe truncated receptor, which lacks nearly all of the N domain, iscapable of mounting a sensitive and robust response to a small agonistligand. The availability of a radioligand that binds to the P1R-delNt,¹²⁵I-[Aib^(1,3),M]PTH(1-21), enabled, for the first time, bindingstudies to be performed on this truncated receptor. Scatchard analysisof our homologous competition binding data yielded Bmax values forP1R-delNt that were not significantly different from those observed forP1R-WT (1.3±0.1 receptors/cell Vs. 1.9±0.8 receptors/cell, respectively,P=0.3). Thus, the truncated receptor is well expressed on the surface ofCOS-7 cells. Not surprisingly, PTH(1-34) failed to inhibit the bindingof ¹²⁵I-[Aib^(1,3),M]PTH(1-21) to P1R-delNt, a result which highlightsthe importance of the interaction between the N domain of the receptorand the C-terminal (15-34) domain of the native peptide in stabilizingthe overall hormone-receptor complex. This result also supports the viewthat the interaction between the amino-terminal portion of PTH and the Jdomain of the receptor is of very weak affinity (Hoare, S. R. J., etal., J. Biol. Chem 276:7741-7753 (2001)). Clearly, the affinity of theinteraction can be improved considerably, as the apparent affinity withwhich [Aib^(1,3),M]PTH(1-14) bound to P1R-delNt (IC₅₀ ˜1,500 nM) wasmuch greater than that of native PTH(1-14), which failed to inhibittracer binding. The 50-fold difference that we observed in theaffinities with which [Aib^(1,3),M]PTH(1-14) and [Aib^(1,3),M]PTH(1-21)bound to P1R-delNt shows that residues C-terminal of residue 14 (e.g.,residues 15-21) contribute binding interactions to the J domain of thereceptor. Studies on related analogs suggest that at least some of thiseffect involves residue 19.

In summary, highly potent PTH(1-14) analogs are obtained by introducingthe conformationally constrained amino acid, Aib, at the N-terminus ofthe peptide. The propensity of Aib to stabilize α-helical structure, andthe high potency with which the modified analogs activated P1R-delNt,show that the N-terminal portion of PTH is α-helical when it is bound tothe activation domain of the receptor. The results also establish thatthe activation domain of the PTH-1R, as defined by P1R-delNt, is fullycapable of mediating high affinity and productive interactions with anagonist ligand.

Example 6 PTH(1-34) Derivatives

We have found that Aib substitutions at positions 1 and 3 in PTH(1-34)([Tyr34]hPTH(1-34)amide) improve cAMP-stimulating potency on P1R-delNTexpressed in COS-7 cells by ˜100-fold, relative to unmodified PTH(1-34)(see Table 5 and FIG. 7B). The Aib substitutions do not detectablyimprove potency of PTH(1-34) on the intact wild-type PTH-1 receptor inCOS-7 cells (Table 5, and FIG. 7A); a result which may be due to thealready maximal response mediated by native PTH(1-34) in these cellswhich express very high levels of the intact receptor. In a lesssensitive cell system, such as with the delNT receptor, in which nearlythe entire amino-terminal extracellular domain of the receptor isdeleted, or perhaps in bone cells in animals expressing low levels ofendogenous PTH receptors, the effect of Aib-1,3 substitutions onPTH(1-34) potency are significant. Peptides with other, above describedmodifications (e.g. Gln10, homoArg11, Ala12, Trp14, Arg19) are much morepotent than PTH(1-34) in COS-7 cells expressing hP1R-delNT as well. Forexample, [Aib^(1,3),Gln¹⁰,Har¹¹, Ala¹²,Trp¹⁴,Arg¹⁹,Tyr³⁴]hPTH(1-34) hasan EC50 value of 1.9±0.6 nM on P1R-delNT. It is expected that the abovedescribed modifications will also be much more potent than PTH(1-34) inother native bone cell systems of low sensitivity. TABLE 5 cAMPResponses of hPTH(1-34) Analogs in COS-7 Cells EC50(nM) EC50(nM) peptidehP1R-WT hP1R-delNt [Tyr³⁴]-hPTH(1-34) 0.44 ± 0.02 2,800 ± 300  [Aib^(1,3), Tyr³⁴]-hPTH(1-34) 0.67 ± 0.18 43 ± 24

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that same can beperformed by modifying or changing the invention with a wide andequivalent range of conditions, formulations and other parametersthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned hereinaboveare herein incorporated in their entirety and by reference to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference.

1. A biologically active peptide consisting essentially of the formulaselected from: (a) X₀₁ValX₀₂GluIleGlnLeuMetHisX₀₃X₀₄X₀₅X₀₅X₀₆X₀₇ (SEQ IDNO. 1); (b) fragments thereof, containing amino acids 1-11, 1-12 or1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C-derivatives thereof; wherein: X₀₁ is an α-helix-stabilizing residue,desaminoGly, desaminoSer or desaminoAla; X₀₂ is an α-helix-stabilizingresidue, Ala, or Ser; X₀₃ is Ala, Gln or Asn; X₀₄ is Arg, Har or Leu;X₀₅ is an α-helix-stabilizing residue, Ala or Gly; X₀₆ is anα-helix-stabilizing residue or Lys; X₀₇ is an α-helix-stabilizingresidue, Trp or His; and wherein at least one of X₀₁, X₀₂, X₀₅, X₀₆ orX₀₇ is an α-helix-stabilizing residue.
 2. The peptide of claim 1,wherein said α-helix-stabilizing amino acid is selected from the groupconsisting of Aib, ACPC (1-aminocyclopropylcarboxylic acid), DEG(diethylglycine) and 1-aminocyclopentanecarboxylic acid.
 3. The peptideof claim 1, wherein said peptide is selected from: (a)AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 2); (b) fragmentsthereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceuticallyacceptable salts thereof; or (d) N- or C- derivatives thereof.
 4. Thepeptide of claim 1, wherein said peptide is selected from: (a)desaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLys His (SEQ ID NO. 3); (b)fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C- derivativesthereof.
 5. The peptide of claim 1, wherein said peptide is selectedfrom: (a) desaminoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLys His (SEQ IDNO. 4); (b) fragments thereof, containing amino acids 1-11, 1-12 or1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C-derivatives thereof.
 6. The peptide of claim 1, wherein said peptide isselected from: (a) desaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLys His(SEQ ID NO. 5); (b) fragments thereof, containing amino acids 1-11, 1-12or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C-derivatives thereof.
 7. The peptide of claim 1, wherein said peptide isselected from: (a) AibValAibGluIleGlnLeuMetHisGlnHarGlyLysTrp (SEQ IDNO. 6); (b) fragments thereof, containing amino acids 1-11, 1-12 or1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C-derivatives thereof.
 8. The peptide of claim 1, said peptide selectedfrom: (a) AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 7); (b)fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C- derivativesthereof.
 9. The peptide of claim 1, said peptide selected from: (a)AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 9); (b) fragmentsthereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceuticallyacceptable salts thereof; or (d) N- or C- derivatives thereof.
 10. Thepeptide of claim 1, said peptide selected from: (a)AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 10); (b)fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C- derivativesthereof.
 11. The peptide of claim 1, said peptide selected from: (a)SerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 11); (b)fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c)pharmaceutically acceptable salts thereof; or (d) N- or C- derivativesthereof.
 12. A biologically active peptide consisting essentially of theformula selected from: (a)AibValAibGluIleGlnLeuNleHisGlnHarAlaLysTrpLeuAla SerValArgArgTyr (SEQ IDNO. 8); (b) fragments thereof, containing amino acids 1-20, 1-19, 1-18,1-17, 1-16 or 1-15; (c) pharmaceutically acceptable salts thereof; or(d) N- or C- derivatives thereof.
 13. The peptide of claim 1, whereinsaid peptide is AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO.2).
 14. The peptide of claim 1, wherein said peptide isdesaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 3). 15.The peptide of claim 1, wherein said peptide isdesamninoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 4). 16.The peptide of claim 1, wherein said peptide isdesaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 5). 17.The peptide of claim 1, wherein said peptide is AibValAibGluIleGlnLeuMetHisGlnHarGlyLysTrp (SEQ ID NO. 6).
 18. The peptide of claim 1,wherein said peptide is AibValAibGluIle GlnLeuMetHisAsnLeuGlyLysHis (SEQID NO. 7).
 19. The peptide of claim 12, wherein said peptide isAibValAibGluIle GlnLeuNleHisGlnHarGlyLysTrpLeuAlaSerValArgArgTyr (SEQ IDNO. 8).
 20. The peptide of claim 1, wherein said peptide isAibValAlaGluIle GlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 9).
 21. Thepeptide of claim 1, wherein said peptide is AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 10).
 22. The peptide of claim 1,wherein said peptide is SerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQID NO. 11).
 23. The peptide of claim 1 or 12, wherein said peptide islabeled.
 24. The peptide of claim 23, wherein said peptide is labeledwith a fluorescent label.
 25. The peptide of claim 23, wherein saidpeptide is labeled with a chemiluminescent label.
 26. The peptide ofclaim 23, wherein said peptide is labeled with a bioluminescent label.27. The peptide of claim 23, wherein said peptide is labeled with aradioactive label.
 28. The peptide of claim 27, wherein said peptide islabeled with ¹²⁵I.
 29. The peptide of claim 27, wherein said peptide islabeled with ^(99m)Tc.
 30. A pharmaceutical composition comprising thebiologically active peptide of claim 1 or 12, and a pharmaceuticallyacceptable carrier.
 31. A method for treating mammalian conditionscharacterized by decreases in bone mass, said method comprisingadministering to a subject in need thereof an effective bonemass-increasing amount of a biologically active peptide of claim 1 or12.
 32. A method for treating mammalian conditions characterized bydecreases in bone mass, said method comprising administering to asubject in need thereof an effective bone mass-increasing amount of acomposition comprising a biologically active peptide of claim 1 or 12and a pharmaceutically acceptable carrier.
 33. A method for determiningrates of bone reformation, bone resorption and/or bone remodelingcomprising administering to a patient an effective amount of a peptideof claim 1 or 12 and determining the uptake of said peptide into thebone of said patient.
 34. The method of claim 32, wherein said conditionto be treated is osteoporosis.
 35. The method of claim 32, wherein saidcondition to be treated is old age osteoporosis.
 36. The method of claim32, wherein said condition to be treated is post-menopausalosteoporosis.
 37. The method of claim 32, wherein said effective amountof said peptide for increasing bone mass is from about 0.01 μg/kg/day toabout 1.0 μg/kg/day.
 38. The method of claim 32, wherein the method ofadministration is parenteral.
 39. The method of claim 32, wherein themethod of administration is subcutaneous.
 40. The method of claim 32,wherein the method of administration is nasal insufflation.
 41. A methodof making the peptide of claim 1 or 12, wherein said peptide issynthesized by solid phase synthesis.
 42. The method of making thepeptide of claim 1 or 12, wherein said peptide is protected by FMOC. 43.The peptide of claim 2, wherein said α-helix-stabilizing amino acid isAib.
 44. A biologically active peptide consisting essentially of theformula selected from: (a) X₀₁ValX₀₂GluIleGlnLeuX₀₃HisX04X₀₅X06X₀₇X₀₈LeuX₀₉Ser X₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsn X₁₄ (SEQ ID NO. 19); (b) pharmaceuticallyacceptable salts thereof; or (c) N- or C- derivatives thereof; wherein:X₀₁ is an α-helix-stabilizing residue desaminoGly, desaminoSer ordesaminoAla; X₀₂ is an α-helix-stabilizing residue, Ala, or Ser; X₀₃ isMet or Nle; X₀₄ is Ala, Gln or Asn; X₀₅ is Arg, Har or Leu; X₀₆ is anα-helix-stabilizing residue, Ala or Gly; X₀₇ is an α-helix-stabilizingresidue or Lys; X₀₈ is an α-helix-stabilizing residue, Trp or His; X₀₉is Ala or Asn; X₁₀ is Met or Val; X₁₁ is Arg or Glu; X₁₂ is Met or Val;X₁₃ is Gln or Glu; X₁₄ is Tyr or Phe; and wherein at least one of X₀₁,X₀₂, X₀₆, X₀₇or X₀₈ is an α-helix-stabilizing residue.
 45. The peptideof claim 44, wherein said α-helix-stabilizing amino acid is selectedfrom the group consisting of Aib, ACPC (1-aminocyclopropylcarboxylicacid), DEG (diethylglycine) and 1-aminocyclopentanecarboxylic acid. 46.The peptide of claim 44, wherein said peptide is selected from: (a)AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉ SerX₁₀X₁₁ArgX₁₂X₁₃ TrpLeuArgLysLysLeuGlnAspValHis AsnX₁₄ (SEQ ID NO. 20); (b) pharmaceuticallyacceptable salts thereof; or (c) N- or C- derivatives thereof.
 47. Thepeptide of claim 44, wherein said peptide is selected from: (a)desaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGln AspValHisAsnX₁₄ (SEQ IDNO. 21); (b) pharmaceutically acceptable salts thereof; or (c) N- or C-derivatives thereof.
 48. The peptide of claim 44, wherein said peptideis selected from: (a) desaminoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGln AspValHisAsnX₁₄ (SEQ IDNO. 22); (b) pharmaceutically acceptable salts thereof; or (c) N- or C-derivatives thereof.
 49. The peptide of claim 44, wherein said peptideis selected from: (a) desaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGln AspValHisAsnX₁₄ (SEQID NO. 23); (b) pharmaceutically acceptable salts thereof; or (c) N- orC- derivatives thereof.
 50. The peptide of claim 44, wherein saidpeptide is selected from: (a)AibValAibGluIleGlnLeuMetHisGlnHarGlyLysTrpLeuX₀₉ SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHis AsnX₁₄ (SEQ ID NO. 24); (b) pharmaceuticallyacceptable salts thereof; or (c) N- or C- derivatives thereof.
 51. Thepeptide of claim 44, said peptide selected from: (a)AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉ SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHis AsnX₁₄ (SEQ ID NO. 25); (b) pharmaceuticallyacceptable salts thereof; or (c) N- or C- derivatives thereof.
 52. Thepeptide of claim 44, said peptide selected from: (a)AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHis AsnX₁₄ (SEQ ID NO. 26); (b) pharmaceuticallyacceptable salts thereof; or (c) N- or C- derivatives thereof.
 53. Thepeptide of claim 44, said peptide selected from: (a)AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX₀₉ SerX_(X)₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHis AsnX₁₄ (SEQ ID NO. 27); (b)pharmaceutically acceptable salts thereof; or (c) N- or C- derivativesthereof.
 54. The peptide of claim 44, said peptide selected from: (a)SerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX₀₉ SerX₁₀X₁₀ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHis AsnX₁₄ (SEQ ID NO. 28); (b) pharmaceuticallyacceptable salts thereof; or (c) N- or C- derivatives thereof.
 55. Abiologically active peptide consisting essentially of the formulaselected from: (a) AibValAibGluIleGlnLeuNleHisGlnHarAlaLysTrpLeuAlaSerValArgArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHis AsnX₁₄ (SEQ ID NO.29); (b) pharmaceutically acceptable salts thereof; or (c) N- or C-derivatives thereof; wherein X₁₂ is Met or Val; X₁₃ is Gln or Glu; andX₁₄ is Tyr or Phe.
 56. The peptide of claim 44, wherein said peptide isAibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 20).
 57. The peptide of claim 44,wherein said peptide isdesaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 21).
 58. The peptide ofclaim 44, wherein said peptide isdesaminoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 22).
 59. The peptide ofclaim 44, wherein said peptide isdesaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉Ser₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 23).
 60. The peptideof claim 44, wherein said peptide is AibValAibGluIleGlnLeuMetHisGlnHarGlyLysTrpLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃TrpLeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 24).
 61. The peptide of claim 44,wherein said peptide is AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 25).
 62. The peptide of claim 44,wherein said peptide is AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 26).
 63. The peptide of claim 44,wherein said peptide is AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 27).
 64. The peptide of claim 44,wherein said peptide is SerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX₀₉SerX₁₀X₁₁ArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 28).
 65. The peptide of claim 55,wherein said peptide is AibValAibGluIleGlnLeuNleHisGlnHarGlyLysTrpLeuAlaSerValArgArgX₁₂X₁₃Trp LeuArgLysLysLeuGlnAspValHisAsnX₁₄ (SEQ ID NO. 29).
 66. The peptide of claim 44 or55, wherein said peptide is labeled.
 67. The peptide of claim 66,wherein said peptide is labeled with a fluorescent label.
 68. Thepeptide of claim 66, wherein said peptide is labeled with achemiluminescent label.
 69. The peptide of claim 66, wherein saidpeptide is labeled with a bioluminescent label.
 70. The peptide of claim66, wherein said peptide is labeled with a radioactive label.
 71. Thepeptide of claim 70, wherein said peptide is labeled with ¹²⁵I.
 72. Thepeptide of claim 70, wherein said peptide is labeled with ^(99m)Tc. 73.A pharmaceutical composition comprising the biologically active peptideof claim 44 or 55, and a pharmaceutically acceptable carrier.
 74. Amethod for treating mammalian conditions characterized by decreases inbone mass, said method comprising administering to a subject in needthereof an effective bone mass-increasing amount of a biologicallyactive peptide of claim 44 or
 55. 75. A method for treating mammalianconditions characterized by decreases in bone mass, said methodcomprising administering to a subject in need thereof an effective bonemass-increasing amount of a composition comprising a biologically activepeptide of claim 44 or 55 and a pharmaceutically acceptable carrier. 76.A method for determining rates of bone reformation, bone resorptionand/or bone remodeling comprising administering to a patient aneffective amount of a peptide of claim 44 or 55 and determining theuptake of said peptide into the bone of said patient.
 77. The method ofclaim 75, wherein said condition to be treated is osteoporosis.
 78. Themethod of claim 75, wherein said condition to be treated is old ageosteoporosis.
 79. The method of claim 75, wherein said condition to betreated is post-menopausal osteoporosis.
 80. The method of claim 75,wherein said effective amount of said peptide for increasing bone massis from about 0.01 μg/kg/day to about 1.0 μg/kg/day.
 81. The method ofclaim 75, wherein the method of administration is parenteral.
 82. Themethod of claim 75, wherein the method of administration issubcutaneous.
 83. The method of claim 75, wherein the method ofadministration is nasal insufflation.
 84. A method of making the peptideof claim 44 or 55, wherein said peptide is synthesized by solid phasesynthesis.
 85. The method of making the peptide of claim 44 or 55,wherein said peptide is protected by FMOC.
 86. The peptide of claim 45,wherein said α-helix-stabilizing amino acid is Aib.